Il-31 dog pruritus model

ABSTRACT

An IL-31 dog pruritus model is provided. This model comprises administering canine IL-31 to dogs to produce a pruritic response; quantitatively measuring pruritic responses in the dogs which were administered canine IL-31; administering a candidate dog IL-31 inhibitor; and assessing the effectiveness of the candidate dog IL-31 inhibitor in reducing prurtic behavior in the treated dogs by challenging the dogs with canine IL-31 following the administration of the candidate dog IL-31 inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/302,730, filed Jun. 12, 2014, which is a continuation of U.S.application Ser. No. 13/536,081, filed Jun. 28, 2012 (now U.S. Pat. No.8,790,651), which claims the benefit of U.S. Provisional Application No.61/510,268, filed Jul. 21, 2011, the contents each of which areincorporated herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to an IL-31 dog pruritus model for use inassessing the effectiveness of an IL-31 inhibitor candidate in reducingprurtic behavior in the treated dogs. The invention also relates to thefield of recombinant monoclonal antibodies and their uses in clinicaland scientific procedures, including diagnostic procedures.

BACKGROUND OF THE INVENTION

Atopic dermatitis has been defined by the American College of VeterinaryDermatology task force as “a genetically-predisposed inflammatory andpruritic allergic skin disease with characteristic clinical features”(Olivry, et al. Veterinary Immunology and Immunopathology 2001;81:143-146). The task force also recognized that the disease in canineshas been associated with allergen-specific IgE (Olivry, et al. 2001supra; Marsella & Olivry Clinics in Dermatology 2003; 21:122-133).Severe pruritus, along with secondary alopecia and erythema, are themost noticeable and concerning symptoms to pet owners.

The prevalence of atopic dermatitis is not known with precision due topoor and inconsistent epidemiological data, but is estimated to be 10%of the total canine population (Marsella & Olivry 2003 supra; Scott, etal. Canadian Veterinary Journal 2002; 43:601-603; Hillier VeterinaryImmunology and Immunopathology 2001; 81:147-151). Globally, about 4.5million dogs are affected with this chronic and lifelong condition.Incidence appears to be increasing. Breed and sex predilections havebeen suspected, but may vary greatly depending on geographical region(Hillier, 2001 supra; Picco, et al. Vet Dermatol. 2008; 19:150-155).

The potential factors involved in allergic dermatitis are numerous andpoorly understood. Components in food may trigger atopic dermatitis(Picco, 2008 supra), as well as environmental allergens such as fleas,dust mites, ragweed, plant extracts, etc. Genetic factors also play animportant role. Although there is no confirmed breed predilection, somemode of inheritance is thought to increase predisposition to atopicdermatitis (Sousa & Marsella Veterinary Immunology and Immunopathology2001; 81:153-157; Schwartzman, et al. Clin. Exp. Immunol. 1971;9:549-569.

Interleukin-31 (IL-31) is a cytokine that was cloned in 2004. It ismainly produced by activated T helper (Th)2 cells (Dillon et al. NatImmunol 2004; 5:752-60), but is also produced in mast cells andmacrophages. IL-31 binds a co-receptor composed of IL-31 receptor A(IL-31 RA) and the oncostatin M receptor (OSMR) (Dillon et al. 2004supra and Bilsborough et al. J Allergy Clin Immunol. 2006117(2):418-25). Receptor activation results in phosphorylation of STATthrough JAK receptor(s). Expression of the co-receptor has been shown inmacrophages, keratinocytes and in dorsal root ganglia. Recently, it hasbeen found that IL-31 is involved in dermatitis, pruritic skin lesions,allergy and airway hypersensitivity. See FIG. 1.

Stimulation of T cells with anti-CD3 and anti-CD28 antibodiesimmediately upregulates IL-31 mRNA expression (Dillon et al. 2004supra). Microarray analysis has shown that IL-31 induces certainchemotactic genes, such as CXCL1, CLL17 (thymus and activation-regulatedchemokine [TARC]), CCL19 (macrophage inflammatory protein [MIP]3β),CCL22 (monocyte-derived chemokine [MDC], CCL23 (MIP3), and CCL4 [MIP]β)(Dillon et al. 2004 supra).

Transgenic mice that over-express IL-31 show skin inflammation,pruritis, severe dermatitis, and alopecia (Dillon et al. 2004 supra).Subcutaneous injection of IL-31 into mice triggers infiltration by theinflammatory cells, neutrophils, eosinophils, lymphocytes, andmacrophages, and results in epidermal thickening and dermal acanthosis.In NC/Nga mice, with atopic dermatitis (AD) due to natural causes, IL-31is overexpressed in skin lesions and correlates with pruritus (Takaokaet al. Eur J. Pharmacol. 2005; 516, 180-181; Takaoka et al. Exp.Dermatol. 2006; 15, 161-167). Also, in murine models, IL-31 has beenshown to induce rapid onset pruritus (Raap et al. J Allergy ClinImmunol. 2008; 122(2):421-3)

Further studies have indicated that IL-31 is associated withatopic-dermatitis-induced skin inflammation and pruritus in humans. Inhuman AD patients, the expression of IL-31 mRNA is considerably higherin skin lesions than in non-lesional skin, and the expression innon-lesional skin is greater than that in normal skin from healthypatients (Sonkoly et al. J Allergy Clin Immunol 2006; 117:411-7).Another study has reported that CD45RO+ (memory) cutaneous lymphocyteantigen (CLA)-positive T cells in the skin of AD patients express IL-31mRNA and protein (Bilsborough et al. 2006 supra). It has also beenreported that IL-31 mRNA overexpression in the skin of patients orallergic contact dermatitis is correlated with IL-4 and IL-13 mRNAexpression, but not with interferon (IFN)-γ mRNA expression (Neis et al.J. Allergy Clin. Immunol. 2006; 118, 930-937). Furthermore, IL-31 serumlevels have been shown to be elevated in human patients with chronicspontaneous urticaria and even more so in patients with AD (Raap et al.Exp Dermatol. 2010; 19(5):464-6). Also, a correlation of the severity ofAD with serum IL-31 levels has been observed in humans (Rapp et al. 2008supra). IL-31 secretion has also been shown to be enhanced in mast cellsfollowing IgE cross-linking and as a response to Staphylococcalsuperantigen in atopic individuals. In addition, IL-31 has been shown tostimulate the production of several pro-inflammatory mediators includingIL-6, IL-8, CXCL1, CC17 and multiple metalloproteinases in human colonicmyofibroblasts (Yagi,et al. International Journal of Molecular Medicine2007; 19(6): 941-946.

Type I hypersensitivity against environmental allergens is considered tobe the main mechanism of canine AD, and the levels of Th2-mediatedcytokines, such as IL-4 are increased in the skin lesions of dogs withAD (Nuttall, et al. Vet. Immunol. Immunopathol. 2002; 87, 379-384).Moreover, infiltration by inflammatory cells, lymphocytes andneutrophils, is an important mechanism underlying the aggravation of theskin lesions; the overexpression of chemotactic genes such asCCL17/TARC, CCR4, and CCL28/mucosae-associated epithelial chemokine(MEC) contributes to the aggravation of skin lesions in the dogs with AD(see, Maeda, et al. Vet. Immunol. Immunopathol. 2005; 103, 83-92; Maeda,et al. Vet. Immunol. Immunopathol.2002b; 90, 145-154; and Maeda, et al.J. Vet. Med. Sci. 2008; 70, 51-55).

Recent evidence has suggested that IL-31 might be involved in promotingallergic inflammation and an airway epithelial response characteristicof allergic asthma (Chattopadhyay, et al. J Biol Chem 2007; 282:3014-26;and Wai, et al. Immunology, 2007; 122, 532-541).

These observations support the hypothesis that IL-31 plays a significantrole in both pruritic and allergic conditions. It would be desirable toprovide a therapeutic antibody against IL-31 useful for treating apruritc condition and/or an allergic condition in dogs or cats.

SUMMARY OF THE INVENTION

The present invention provides an IL-31 dog pruritus model comprisingadministering canine IL-31 to dogs to produce a pruritic response;quantitatively measuring pruritic responses in the dogs which wereadministered canine IL-31; administering a candidate dog IL-31inhibitor; and assessing the effectiveness of the candidate dog IL-31inhibitor in reducing prurtic behavior in the treated dogs bychallenging the dogs with canine IL-31 following the administration ofthe candidate dog IL-31 inhibitor.

In one embodiment, the canine IL-31 administered to the dogs to producethe pruritic response is recombinant canine IL-31. In a particularembodiment, the recombinant canine IL-31 has the amino acid sequence ofSEQ ID NO: 32.

In one embodiment, the canine IL-31 is administered parenterally to thedog.

In a further embodiment, the canine IL-31 is administered to the dog ata dose of 1 to 1.5 μg/kg.

In another embodiment, the pruritic response is a transient response. Inone embodiment, the transient pruritic response lasts less than 24hours. In a further embodiment, the pruritic responses in the dogsinduced by canine IL-31 are selected from licking, chewing, scratching,head-shaking, or scooting.

In one embodiment, the pruritic behavior measurements are performedusing real-time video surveillance using a categorical scoring system.In a particular embodiment, at consecutive time intervals, “yes/no”decisions are made in regards to whether puritic behavior was displayedby each dog. In one embodiment, the consecutive time intervals are 1minute intervals. In another embodiment, at the end of a designatedobservation period, the numbers of yes determinations are added togetherto come up with a cumulative Pruritic Score Index (PSI).

In one embodiment, a first PSI measurement is a baseline score measuredimmediately prior to the canine IL-31 challenge. In another embodiment,an additional PSI measurement is determined following the canine IL-31challenge.

In one embodiment, the candidate dog IL-31 inhibitor is an antibody thatspecifically binds to canine IL-31. In another embodiment, the antibodyspecifically binds to a canine IL-31 having the amino acid sequence ofSEQ ID NO: 32. In a further embodiment, the antibody is a monoclonalantibody. In a still further embodiment, the monoclonal antibody iscaninized or felinized.

In yet another embodiment, the candidate dog IL-31 inhibitor isadministered to the dog parenterally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the IL-31 pathway.

FIG. 2 is a schematic representation of the general structure of a mouseimmunoglobulin G (IgG) molecule highlighting the antigen binding site.

FIG. 3 is a schematic representation of the general structure of amouse:canine chimeric IgG

FIG. 4 is an illustration showing speciation or “caninization” of amouse IgG, mouse CDRs are grafted onto canine frameworks identified fromsequence databases

FIG. 5 is an illustration of a “heterochimeric” monoclonal antibodypairing the chimeric light chain with a fully caninized heavy chain.

FIG. 6 ELISA Titers from IL-31 Immunized Mice (CF-1 MU#1-4) relative topre-bleed and positive control mice.

FIG. 7 is an illustration of antibody variable chains showing primers toconstant regions and degenerate primers directed at mouse variableregions.

FIG. 8 is a graph of the pilot efficacy of chimeric 11E12 in a placebocontrolled, single dose, SC study (76A60).

FIG. 9 is of a table showing the individual pruritic scores from dogsenrolled in study 76A60.

FIG. 10 is of Western blots showing binding of chimeric (Blot #1),caninized (Blot#2), and heterochimeric (Blots #3 and 4) versions of 11E1 2 to canine IL-31. The heterochimera in Blot #3 has a caninized lightchain paired with a chimeric heavy chain. The heterochimera in Blot #4has the chimeric light chain paired with the caninized heavy chain. Eachnitrocellulose blot contains-left lane, pre-stained protein standards(Seeblue plus 2, Invitrogen Corp., Carlsbad, Calif.) and right lane, 800ng of canine IL-31.

FIG. 11 is a schematic overview of caninized 11E1 2 light chainframework substitution work. The sequence designated as “11E12_VI”corresponds to SEQ ID NO: 19; the sequence designated as“11E12_VI_gapped” corresponds to SEQ ID NO: 19 except that it is missingthe CDR sequences corresponding to RASES (SEQ ID NO: 10), RASNLES (SEQID NO: 13) and QQSNKDPLT (SEQ ID NO: 16); the sequence designated as“11E12_VL_cUn.1” corresponds to SEQ ID NO: 79; and the sequencedesignated as “Consensus” corresponds to SEQ ID NO: 80.

FIG. 12 is of Western blots showing binding of caninized versions of11E12 with single backmutations to mouse framework 2 light chainresidues. Each nitrocellulose blot contains- left lane, pre-stainedprotein standards (Seeblue plus 2, Invitrogen Corp., Carlsbad, Calif.)and right lane, 800 ng of canine IL-31.

FIG. 13 is of Western blots with full length and truncated canine IL-31proteins. Individual nitrocellulose blots were probed with A) anti-HisB) 34D03 and C)11E12 antibodies. Lanes 1-9 of the blots correspond tothe following: Lane 1-pre-stained protein standards (Seeblue plus 2,Invitrogen Corp., Carlsbad, Calif.); Lane 2-full-length canine IL-31;Lane 3-N-terminal truncation-20N; Lane 4-N-terminal truncation −40N;Lane 5-N-terminal truncation −60N; Lane 6-C-terminal truncation −20C;Lane 7-C-terminal truncation −40C; Lane 8-C-terminal truncation −60C;and Lane 9-beta-galactosidase (lacZ). Note: full length IL-31 andproteins with C-terminal truncations (−20, −40C, and −60C) showed nodetectable expression under these conditions.

FIG. 14 is of Western blots with truncated canine IL-31 proteins.Individual nitrocellulose blots were probed with A) anti-His B) 11E12and C) 34D03 antibodies. Lanes 1-5 of the blots correspond to thefollowing: Lane 1-pre-stained protein standards (Seeblue plus 2,Invitrogen Corp., Carlsbad, Calif.); Lane 2-C-terminal truncations atpositions 20-122; Lane 3-C-terminal truncations at positions 20-100;Lane 4-C-terminal truncations at positions 20-80; and Lane5-beta-galactosidase (lacZ).

FIG. 15 is a section of Western blots with lysates of E. coli strainsexpressing canine IL-31 with alanine substituted for each amino acidposition (76-122). Individual nitrocellulose blots were probed withanti-His, 11E12 and, 34D03 antibodies, as shown in the Figure.

FIG. 16 is a section of Western blots with double and triple mutationsin canine IL-31. −20N protein lysate was run as a positive control.

FIG. 17 is a graph showing the pruritic scores for dogs injectedsubcutaneously with caninized 34D03 antibody (1.0 mg/kg). Pruriticscores were measured on each study day prior to (baseline response) andfollowing (2 h response) challenge with 1.5 μg/kg canine IL-31.

FIG. 18 is a 4-12% Bis Tris SDS PAGE with purified canine and felineIL-31 proteins. Panel A shows coomassie staining of proteins run underreducing conditions. Panel B shows coomassie staining of proteins rununder non-reducing conditions Panels. Panels C and D are the Westernblots of gels identical to A and B respectively, probed with an anti-Hisantibody. Lane 1-canine IL-31; Lane 2-feline IL-31; Lane 3-pre-stainedprotein standards (Seeblue plus 2, Invitrogen Corp., Carlsbad, Calif.);Lane 4-canine IL-31; and Lane 5-feline IL-31.

FIG. 19 is a graph of pSTAT signaling in canine DH-82 monocytes inducedby canine and feline IL-31 produced in E. coli. Canine IL-31 (CHO) isthe reference protein used for all previous cell-based assays, dogpruritus model, and as the immunogen for initial identification ofantibodies.

FIG. 20 is an alignment showing the sequence conservation between felineand canine IL-31 in the region of the protein involved in binding of11E12 and 34D03 antibodies (annotated with a plus sign). The canineIL-31 sequence corresponds to amino acid residues 98 to 113 of SEQ IDNO: 32; and the feline IL-31 sequence corresponds to amino acid residues87 to 102 of SEQ ID NO: 70.

FIG. 21 is of Western blots with IL-31 proteins. Individualnitrocellulose blots were probed with A) anti-His B) 11E12 and C) 34D03antibodies. Note-Canine IL-31 (CHO) does not contain a 6-His tag.

FIG. 22 is a graph showing the inhibition of canine IL-31 induced pSTATsignaling in canine DH82 monocytes comparing felinized and caninizedantibody 34D03.

FIG. 23 is a Western blot of feline and canine IL-31 under reducingconditions probed with felinized antibody 34D03.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a variable heavy chain CDR1 referred to herein as11E12-VH-CDR1;

SEQ ID NO: 2 is a variable heavy chain CDR1 referred to herein as19D07-VH-CDR1;

SEQ ID NO: 3 is a variable heavy chain CDR1 referred to herein as34D03-VH-CDR1;

SEQ ID NO: 4 is a variable heavy chain CDR2 referred to herein as11E12-VH-CDR2;

SEQ ID NO: 5 is a variable heavy chain CDR2 referred to herein as19D07-VH-CDR2;

SEQ ID NO: 6 is a variable heavy chain CDR2 referred to herein as34D03-VH-CDR2;

SEQ ID NO: 7 is a variable heavy chain CDR3 referred to herein as11E12-VH-CDR3;

SEQ ID NO: 8 is a variable heavy chain CDR3 referred to herein as19D07-VH-CDR3;

SEQ ID NO: 9 is a variable heavy chain CDR3 referred to herein as34D03-VH-CDR3;

SEQ ID NO: 10 is a variable light chain CDR1 referred to herein as11E12-VL-CDR1;

SEQ ID NO: 11 is a variable light chain CDR1 referred to herein as19D07-VL-CDR1;

SEQ ID NO: 12 is a variable light chain CDR1 referred to herein as34D03-VL-CDR1;

SEQ ID NO: 13 is a variable light chain CDR2 referred to herein as11E12-VL-CDR2;

SEQ ID NO: 14 is a variable light chain CDR2 referred to herein as19D07-VL-CDR2;

SEQ ID NO: 15 is a variable light chain CDR2 referred to herein as34D03-VL-CDR2;

SEQ ID NO: 16 is a variable light chain CDR2 referred to herein as11E12-VL-CDR3;

SEQ ID NO: 17 is a variable light chain CDR3 referred to herein as19D07-VL-CDR3;

SEQ ID NO: 18 is a variable light chain CDR3 referred to herein as34D03-VL-CDR3;

SEQ ID NO: 19 is a variable light chain sequence referred to herein asMU-11E12-VL;

SEQ ID NO: 20 is a variable light chain sequence referred to herein asCAN-11E1 2-VL-cUn-FW2;

SEQ ID NO: 21 is a variable light chain sequence referred to herein asCAN-11E12-VL-cUn-13;

SEQ ID NO: 22 is a variable light chain sequence referred to herein asMU-19D07-VL;

SEQ ID NO: 23 is a variable light chain sequence referred to herein asCAN-19D07-VL-998-1;

SEQ ID NO: 24 is a variable light chain sequence referred to herein asMU-34D03-VL;

SEQ ID NO: 25 is a variable light chain sequence referred to herein asCAN-34D03-VL-998-1;

SEQ ID NO: 26 is a variable heavy chain sequence referred to herein asMU-11E12-VH;

SEQ ID NO: 27 is a variable heavy chain sequence referred to herein asCAN-11E12-VH-415-1;

SEQ ID NO: 28 is a variable heavy chain sequence referred to herein asMU-19D07-VH;

SEQ ID NO: 29 is a variable heavy chain sequence referred to herein asCAN-19D07-VH-400-1;

SEQ ID NO: 30 is a variable heavy chain sequence referred to herein asMU-34D03-VH;

SEQ ID NO: 31 is a variable heavy chain sequence referred to herein asCAN-34D03-VH-568-1;

SEQ ID NO: 32 is the amino acid sequence corresponding to GenBankAccession No. C7G0W1 and corresponds to Canine IL-31 full-lengthprotein;

SEQ ID NO: 33 is the nucleotide sequence corresponding to GenBankAccession No. C7G0W1 and corresponds to the nucleotide sequence encodingCanine IL-31 full-length protein;

SEQ ID NO: 34 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as MU-11E12-VL;

SEQ ID NO: 35 is the nucleotide sequence encoding the variable heavychain sequence referred to herein as MU-11E12-VH;

SEQ ID NO: 36 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as MU-19D07-VL;

SEQ ID NO: 37 is the nucleotide sequence encoding the variable heavychain sequence referred to herein as MU-19D07-VH;

SEQ ID NO: 38 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as MU-34D03-VL;

SEQ ID NO: 39 is the nucleotide sequence encoding the variable heavychain sequence referred to herein as MU-34D03-VH;

SEQ ID NO: 40 is the amino acid sequence for the canine heavy chainconstant region referred to herein as HC-64 (GenBank accession no.AF354264);

SEQ ID NO: 41 is the nucleotide sequence encoding the canine heavy chainconstant region referred to herein as HC-64 (GenBank accession no.AF354264);

SEQ ID NO: 42 is the amino acid sequence for the canine heavy chainconstant region referred to herein as HC-65 (GenBank accession no.AF354265);

SEQ ID NO: 43 is the nucleotide sequence encoding the canine heavy chainconstant region referred to herein as HC-65 (GenBank accession no.AF354265);

SEQ ID NO: 44 is the amino acid sequence for the canine light chainconstant region referred to herein as kappa (GenBank Accession No.XP_532962);

SEQ ID NO: 45 is the nucleotide sequence encoding the canine light chainconstant region referred to as kappa (GenBank Accession No. XP_532962);

SEQ ID NO: 46 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as CAN-19D07-VL-998-1;

SEQ ID NO: 47 is the nucleotide sequence encoding the variable heavychain sequence referred to herein as CAN-19D07-VH-998-1;

SEQ ID NO: 48 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as CAN-34D03-VL-998-1;

SEQ ID NO: 49 is the nucleotide sequence encoding the variable heavychain sequence referred to herein as CAN-34D03-VH-568-1;

SEQ ID NO: 50 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as CAN-11E12-VL-cUn-FW2;

SEQ ID NO: 51 is the nucleotide sequence encoding the variable heavychain sequence referred to herein as CAN-11E12-VH-415-1;

SEQ ID NO: 52 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as CAN-11E12-VL-cUn-13;

SEQ ID NO: 53 is a variable light chain sequence referred to herein asCAN-11E12_VL_cUn_1;

SEQ ID NO: 54 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as CAN-11E12-VL-cUn-1;

SEQ ID NO: 55 corresponds to the amino acid sequence of the canine IL-31full-length construct used herein for E. coli expression;

SEQ ID NO: 56 is the nucleotide sequence corresponding to the canineIL-31 full-length construct used herein for E. coli expression;

SEQ ID NO: 57 is the amino acid sequence of the canine IL-31 -20Nconstruct for E. coli expression;

SEQ ID NO: 58 is the nucleotide sequence corresponding to the canineIL-31 -20N construct for E. coli expression;

SEQ ID NO: 59 is the amino acid sequence of the canine IL-31 -40Nconstruct for E. coli expression;

SEQ ID NO: 60 is the nucleotide sequence corresponding to the canineIL-31 -40N construct for E. coli expression;

SEQ ID NO: 61 is the amino acid sequence of the canine IL-31 -60Nconstruct for E. coli expression;

SEQ ID NO: 62 is the nucleotide sequence corresponding to the canineIL-31 -60N construct for E. coli expression;

SEQ ID NO: 63 is the amino acid sequence of the canine IL-31 20-122construct for E. coli expression;

SEQ ID NO: 64 is the nucleotide sequence corresponding to the canineIL-31 20-122 construct for E. coli expression;

SEQ ID NO: 65 is the amino acid sequence of the canine IL-31 20-100construct for E. coli expression;

SEQ ID NO: 66 is the nucleotide sequence corresponding to the canineIL-31 20-100 construct for E. coli expression;

SEQ ID NO: 67 is the is the amino acid sequence of the canine IL-3120-80 construct for E. coli expression;

SEQ ID NO: 68 is the nucleotide sequence corresponding to the canineIL-31 20-80 construct for E. coli expression;

SEQ ID NO: 69 is the nucleotide sequence corresponding to the felineIL-31 full-length construct for E. coli expression;

SEQ ID NO: 70 is the amino acid sequence corresponding to the felineIL-31 full-length construct for E. coli expression;

SEQ ID NO: 71 is a variable light chain sequence referred to herein asFEL-34D03-VL-021-1;

SEQ ID NO: 72 is the nucleotide sequence encoding the variable lightchain sequence referred to herein as FEL-34D03-VL-021-1;

SEQ ID NO: 73 is a variable heavy chain sequence referred to herein asFEL-34D03-VH-035-1;

SEQ ID NO: 74 is the nucleotide sequence encoding the variable heavychain sequence referred to herein as FEL-34D03-VH-035-1;

SEQ ID NO: 75 is the amino acid sequence for the feline heavy chainconstant region referred to herein as HC-A Feline (GenBank accession no.AB016710.1);

SEQ ID NO: 76 is the nucleotide sequence encoding the feline heavy chainconstant region referred to herein as HC-A Feline (GenBank accession no.AB016710.1);

SEQ ID NO: 77 is the amino acid sequence for the feline light chainconstant region referred to herein as LC-Kappa Feline (GenBank accessionno. AF198257.1);

SEQ ID NO: 78 is the nucleotide sequence encoding the feline light chainconstant region referred to herein as LC-Kappa Feline (GenBank accessionno. AF198257.1);

SEQ ID NO: 79 is a variable light chain sequence referred to as11E12_VL_cUn.1 in FIG. 11.

SEQ ID NO: 80 is a variable light chain sequence referred to asConsensus in FIG. 11.

Definitions

Before describing the present invention in detail, several terms used inthe context of the present invention will be defined. In addition tothese terms, others are defined elsewhere in the specification, asnecessary. Unless otherwise expressly defined herein, terms of art usedin this specification will have their art-recognized meanings.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, reference to “an antibody” includes a pluralityof such antibodies.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers.

Epitope, as used herein, refers to the antigenic determinant recognizedby the CDRs of the antibody. In other words, epitope refers to thatportion of any molecule capable of being recognized by, and bound by, anantibody. Unless indicated otherwise, the term “epitope” as used herein,refers to the region of IL-31 to which an anti-IL-31 agent is reactiveto.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of being recognizedby, and bound by, an antibody (the corresponding antibody binding regionmay be referred to as a paratope). In general, epitopes consist ofchemically active surface groupings of molecules, for example, aminoacids or sugar side chains, and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.

The term “specifically” in the context of antibody binding, refers tohigh avidity and/or high affinity binding of an antibody to a specificantigen, i.e., a polypeptide, or epitope. In many embodiments, thespecific antigen is an antigen (or a fragment or subfraction of anantigen) used to immunize the animal host from which theantibody-producing cells were isolated. Antibody specifically binding anantigen is stronger than binding of the same antibody to other antigens.Antibodies which bind specifically to a polypeptide may be capable ofbinding other polypeptides at a weak, yet detectable level (e.g., 10% orless of the binding shown to the polypeptide of interest). Such weakbinding, or background binding, is readily discernible from the specificantibody binding to a subject polypeptide, e.g. by use of appropriatecontrols. In general, specific antibodies bind to an antigen with abinding affinity with a KD of 10⁻⁷ M or less, e.g., 10⁻⁸ M or less(e.g., 10⁻⁸ M or less, 10⁻¹⁰ or less, 10⁻¹¹ or less, 10⁻¹² or less, or10⁻¹³ or less, etc.).

As used herein, the term “antibody” refers to an intact immunoglobulinhaving two light and two heavy chains. Thus a single isolated antibodyor fragment may be a polyclonal antibody, a monoclonal antibody, asynthetic antibody, a recombinant antibody, a chimeric antibody, aheterochimeric antibody, a caninized antibody, or a felinized antibody.The term “antibody” preferably refers to monoclonal antibodies andfragments thereof, and immunologic binding equivalents thereof that canbind to the IL-31 protein and fragments thereof. The term antibody isused both to refer to a homogeneous molecular, or a mixture such as aserum product made up of a plurality of different molecular entities.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 Daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains. FIG. 2 isan example of the general structure of a native mouse immunoglobulin G(IgG) highlighting the antigen binding site.

The term “antibody fragment” refers to less than an intact antibodystructure, including, without limitation, an isolated single antibodychain, an Fv construct, a Fab construct, an Fc construct, a light chainvariable or complementarity determining region (CDR) sequence, etc.

The term “variable” region comprises framework and CDRs (otherwise knownas hypervariables) and refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework region (FR). The variabledomains of native heavy and light chains each comprise multiple FRs,largely adopting a β-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the a-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see Kabat, et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), pages 647-669). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (Kabat, et al. (1991),above) and/or those residues from a “hypervariable loop” (Chothia andLesk J. Mol. Biol. 196:901-917 (1987). “Framework” or “FR” residues arethose variable domain residues other than the hypervariable regionresidues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.Presently there are five major classes of immunoglobulins: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 (asdefined by mouse and human designation). The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known in multiple species. The prevalence ofindividual isotypes and functional activities associated with theseconstant domains are species-specific and must be experimentallydefined.

“Monoclonal antibody” as defined herein is an antibody produced by asingle clone of cells (specifically, a single clone of hybridoma cells)and therefore a single pure homogeneous type of antibody. All monoclonalantibodies produced from the same clone are identical and have the sameantigen specificity. The term “monoclonal” pertains to a single clone ofcells, a single cell, and the progeny of that cell.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species, while the remainder ofthe chain(s) is identical with or homologous to corresponding sequencesin antibodies derived from another species, as well as fragments of suchantibodies, so long as they exhibit the desired biological activity.Typically, chimeric antibodies are antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to canine constant segments. FIG. 3 isa schematic representation of the general structure of one embodiment ofa mouse:canine IgG. In this embodiment, the antigen binding site isderived from mouse while the H portion is canine.

“Caninized” forms of non-canine (e.g., murine) antibodies aregenetically engineered antibodies that contain minimal sequence derivedfrom non-canine immunoglobulin. Caninized antibodies are canineimmunoglobulin sequences (recipient antibody) in which hypervariableregion residues of the recipient are replaced by hypervariable regionresidues from a non-canine species (donor antibody) such as mouse havingthe desired specificity, affinity, and capacity. In some instances,framework region (FR) residues of the canine immunoglobulin sequencesare replaced by corresponding non-canine residues. Furthermore,caninized antibodies may include residues that are not found in therecipient antibody or in the donor antibody. These modifications aremade to further refine antibody performance. In general, the caninizedantibody will include substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable regions correspond to those of a non-canine immunoglobulinsequence and all or substantially all of the FRs are those of a canineimmunoglobulin sequence. The caninized antibody optionally also willcomprise a complete, or at least a portion of an immunoglobulin constantregion (Fc), typically that of a canine immunoglobulin sequence. FIG. 4is an illustration of one embodiment showing speciation or caninizationof a mouse IgG. In this embodiment, mouse CDRs are grafted onto canineframeworks.

“Felinized” forms of non-feline (e.g., murine) antibodies aregenetically engineered antibodies that contain minimal sequence derivedfrom non-feline immunoglobulin. Felinized antibodies are felineimmunoglobulin sequences (recipient antibody) in which hypervariableregion residues of the recipient are replaced by hypervariable regionresidues from a non-feline species (donor antibody) such as mouse havingthe desired specificity, affinity, and capacity. In some instances,framework region (FR) residues of the feline immunoglobulin sequencesare replaced by corresponding non-feline residues. Furthermore,felinized antibodies may include residues that are not found in therecipient antibody or in the donor antibody. These modifications aremade to further refine antibody performance. In general, the felinizedantibody will include substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable regions correspond to those of a non-feline immunoglobulinsequence and all or substantially all of the FRs are those of a felineimmunoglobulin sequence. The felinized antibody optionally also willcomprise a complete, or at least a portion of an immunoglobulin constantregion (Fc), typically that of a feline immunoglobulin sequence.

The term “heterochimeric” as defined herein, refers to an antibody inwhich one of the antibody chains (heavy or light) is caninized while theother is chimeric. FIG. 5 depicts one embodiment of a heterochimericmolecule. In this embodiment, a caninized variable heavy chain (whereall of the CDRs are mouse and all FRs are canine) is paired with achimeric variable light chain (where all of the CDRs are mouse and allFRs are mouse. In this embodiment, both the variable heavy and variablelight chains are fused to a canine constant region.

A “variant” anti-IL-31 antibody, refers herein to a molecule whichdiffers in amino acid sequence from a “parent” anti-IL-31 antibody aminoacid sequence by virtue of addition, deletion, and/or substitution ofone or more amino acid residue(s) in the parent antibody sequence andretains at least one desired activity of the parent anti-IL-31-antibody.Desired activities can include the ability to bind the antigenspecifically, the ability to reduce, inhibit or neutralize IL-31activity in an animal, and the ability to inhibit IL-31-mediated pSTATsignaling in a cell-based assay. In one embodiment, the variantcomprises one or more amino acid substitution(s) in one or morehypervariable and/or framework region(s) of the parent antibody. Forexample, the variant may comprise at least one, e.g. from about one toabout ten, and preferably from about two to about five, substitutions inone or more hypervariable and/or framework regions of the parentantibody. Ordinarily, the variant will have an amino acid sequencehaving at least 50% amino acid sequence identity with the parentantibody heavy or light chain variable domain sequences, more preferablyat least 65%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% sequence identity. Identity or homologywith respect to this sequence is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withthe parent antibody residues, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology. The variant retains theability to bind an IL-31 and preferably has desired activities which aresuperior to those of the parent antibody. For example, the variant mayhave a stronger binding affinity, enhanced ability to reduce, inhibit orneutralize IL-31 activity in an animal, and/or enhanced ability toinhibit IL-31-mediated pSTAT signaling in a cell-based assay.

A “variant” nucleic acid, refers herein to a molecule which differs insequence from a “parent” nucleic acid. Polynucleotide sequencedivergence may result from mutational changes such as deletions,substitutions, or additions of one or more nucleotides. Each of thesechanges may occur alone or in combination, one or more times in a givensequence.

The “parent” antibody herein is one that is encoded by an amino acidsequence used for the preparation of the variant. Preferably, the parentantibody has a canine framework region and, if present, has canineantibody constant region(s). For example, the parent antibody may be acaninized or canine antibody. As another example, the parent antibodymay be a felinized or feline antibody. As yet another example, theparent antibody is a murine monoclonal antibody.

The term “isolated” means that the material (e.g., antibody or nucleicacid) is separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials that would interfere with diagnostic or therapeutic uses forthe material, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. With respect to nucleic acid, an isolatednucleic acid may include one that is separated from the 5′ to 3′sequences with which it is normally associated in the chromosome. Inpreferred embodiments, the material will be purified to greater than 95%by weight of the material, and most preferably more than 99% by weight.Isolated material includes the material in situ within recombinant cellssince at least one component of the material's natural environment willnot be present. Ordinarily, however, isolated material will be preparedby at least one purification step.

The word “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to the antibody ornucleic acid. The label may itself be detectable by itself (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable.

The terms “nucleic acid”, “polynucleotide”, “nucleic acid molecule” andthe like may be used interchangeably herein and refer to a series ofnucleotide bases (also called “nucleotides”) in DNA and RNA. The nucleicacid may contain deoxyribonucleotides, ribonucleotides, and/or theiranalogs. The term “nucleic acid” includes, for example, single-strandedand double-stranded molecules. A nucleic acid can be, for example, agene or gene fragment, exons, introns, a DNA molecule (e.g., cDNA), anRNA molecule (e.g., mRNA), recombinant nucleic acids, plasmids, andother vectors, primers and probes. Both 5′ to 3′ (sense) and 3′ to 5′(antisense) polynucleotides are included.

A “subject” or “patient” refers to an animal in need of treatment thatcan be affected by molecules of the invention. Animals that can betreated in accordance with the invention include vertebrates, withmammals such as canine, feline, and equine animals being particularlypreferred examples.

A “therapeutically effective amount” (or “effective amount”) refers toan amount of an active ingredient, e.g., an agent according to theinvention, sufficient to effect beneficial or desired results whenadministered to a subject or patient. An effective amount can beadministered in one or more administrations, applications or dosages. Atherapeutically effective amount of a composition according to theinvention may be readily determined by one of ordinary skill in the art.In the context of this invention, a “therapeutically effective amount”is one that produces an objectively measured change in one or moreparameters associated with treatment of a pruritic condition or anallergic condition including clinical improvement in symptoms. Ofcourse, the therapeutically effective amount will vary depending uponthe particular subject and condition being treated, the weight and ageof the subject, the severity of the disease condition, the particularcompound chosen, the dosing regimen to be followed, timing ofadministration, the manner of administration and the like, all of whichcan readily be determined by one of ordinary skill in the art.

As used herein, the term “therapeutic” encompasses the full spectrum oftreatments for a disease or disorder. A “therapeutic” agent of theinvention may act in a manner that is prophylactic or preventive,including those that incorporate procedures designed to target animalsthat can be identified as being at risk (pharmacogenetics); or in amanner that is ameliorative or curative in nature; or may act to slowthe rate or extent of the progression of at least one symptom of adisease or disorder being treated.

“Treatment”, “treating”, and the like refers to both therapeutictreatment and prophylactic or preventative measures. Animals in need oftreatment include those already with the disorder as well as those inwhich the disorder is to be prevented. The term “treatment” or“treating” of a disease or disorder includes preventing or protectingagainst the disease or disorder (that is, causing the clinical symptomsnot to develop); inhibiting the disease or disorder (i.e., arresting orsuppressing the development of clinical symptoms; and/or relieving thedisease or disorder (i.e., causing the regression of clinical symptoms).As will be appreciated, it is not always possible to distinguish between“preventing” and “suppressing” a disease or disorder since the ultimateinductive event or events may be unknown or latent. Accordingly, theterm “prophylaxis” will be understood to constitute a type of“treatment” that encompasses both “preventing” and “suppressing.” Theterm “treatment” thus includes “prophylaxis”.

The term “allergic condition” is defined herein as a disorder or diseasecaused by an interaction between the immune system and a substanceforeign to the body. This foreign substance is termed “an allergen”.Common allergens include aeroallergens, such as pollens, dust, molds,dust mite proteins, injected saliva from insect bites, etc. Examples ofallergic conditions include, but are not limited to, the following:allergic dermatitis, summer eczema, urticaria, heaves, inflammatoryairway disease, recurrent airway obstruction, airwayhyper-responsiveness, chronic obstructive pulmonary disease, andinflammatory processes resulting from autoimmunity, such as Irritablebowel syndrome (IBS).

The term “pruritic condition” is defined herein as a disease or disordercharacterized by an intense itching sensation that produces the urge torub or scratch the skin to obtain relief. Examples of pruriticconditions include, but are not limited to the following: atopicdermatitis, eczema, psoriasis, scleroderma, and pruritus.

As used herein, the terms “cell”, “cell line”, and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell (e.g., bacterial cells,yeast cells, mammalian cells, and insect cells) whether located in vitroor in vivo. For example, host cells may be located in a transgenicanimal. Host cell can be used as a recipient for vectors and may includeany transformable organism that is capable of replicating a vectorand/or expressing a heterologous nucleic acid encoded by a vector.

A “composition” is intended to mean a combination of active agent andanother compound or composition which can be inert (e.g., a label), oractive, such as an adjuvant.

As defined herein, pharmaceutically acceptable carriers suitable for usein the invention are well known to those of skill in the art. Suchcarriers include, without limitation, water, saline, buffered saline,phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions.Other conventionally employed diluents, adjuvants and excipients, may beadded in accordance with conventional techniques. Such carriers caninclude ethanol, polyols, and suitable mixtures thereof, vegetable oils,and injectable organic esters. Buffers and pH adjusting agents may alsobe employed. Buffers include, without limitation, salts prepared from anorganic acid or base. Representative buffers include, withoutlimitation, organic acid salts, such as salts of citric acid, e.g.,citrates, ascorbic acid, gluconic acid, histidine-HCl, carbonic acid,tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris,trimethanmine hydrochloride, or phosphate buffers. Parenteral carrierscan include sodium chloride solution, Ringer's dextrose, dextrose,trehalose, sucrose, and sodium chloride, lactated Ringer's or fixedoils. Intravenous carriers can include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose andthe like. Preservatives and other additives such as, for example,antimicrobials, antioxidants, chelating agents (e.g., EDTA), inert gasesand the like may also be provided in the pharmaceutical carriers. Thepresent invention is not limited by the selection of the carrier. Thepreparation of these pharmaceutically acceptable compositions, from theabove-described components, having appropriate pH isotonicity, stabilityand other conventional characteristics is within the skill of the art.See, e.g., texts such as Remington: The Science and Practice ofPharmacy, 20th ed, Lippincott Williams & Wilkins, publ., 2000; and TheHandbook of Pharmaceutical Excipients, 4.sup.th edit., eds. R. C. Roweet al, APhA Publications, 2003.

The term “conservative amino acid substitution” indicates any amino acidsubstitution for a given amino acid residue, where the substituteresidue is so chemically similar to that of the given residue that nosubstantial decrease in polypeptide function (e.g., enzymatic activity)results. Conservative amino acid substitutions are commonly known in theart and examples thereof are described, e.g., in U.S. Pat. Nos.6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferredembodiment, a conservative amino acid substitution will be any one thatoccurs within one of the following six groups

-   -   1. Small aliphatic, substantially non-polar residues: Ala, Gly,        Pro, Ser, and Thr;    -   2. Large aliphatic, non-polar residues: Ile, Leu, and Val; Met;    -   3. Polar, negatively charged residues and their amides: Asp and        Glu;    -   4. Amides of polar, negatively charged residues: Asn and Gln;        His;    -   5. Polar, positively charged residues: Arg and Lys; His; and    -   6. Large aromatic residues: Trp and Tyr; Phe.        -   In a preferred embodiment, a conservative amino acid            substitution will be any one of the following, which are            listed as Native Residue (Conservative Substitutions) pairs:            Ala (Ser); Arg (Lys); Asn (Gln; His); Asp (Glu); Gln (Asn);            Glu (Asp); Gly (Pro); His (Asn; Gln); Ile (Leu; Val); Leu            (Ile; Val); Lys (Arg; Gln; Glu); Met (Leu; Ile); Phe (Met;            Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe);            and Val (Ile; Leu).

Just as a polypeptide may contain conservative amino acidsubstitution(s), a polynucleotide hereof may contain conservative codonsubstitution(s). A codon substitution is considered conservative if,when expressed, it produces a conservative amino acid substitution, asdescribed above. Degenerate codon substitution, which results in noamino acid substitution, is also useful in polynucleotides according tothe present invention. Thus, e.g., a polynucleotide encoding a selectedpolypeptide useful in an embodiment of the present invention may bemutated by degenerate codon substitution in order to approximate thecodon usage frequency exhibited by an expression host cell to betransformed therewith, or to otherwise improve the expression thereof.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Unless otherwise defined, scientific and technical terms used inconnection with the antibodies described herein shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art.

Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transfection (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification, See e.g., Sambrook et al. MOLECULAR CLONING: LAB. MANUAL(3rd ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y., 2001)and Ausubel et al. Current Protocols in Molecular Biology (New York:Greene Publishing Association/Wiley Interscience), 1993. Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application.

The present invention provides for recombinant monoclonal antibodies andpeptides and their uses in clinical and scientific procedures, includingdiagnostic procedures. With the advent of methods of molecular biologyand recombinant technology, it is possible to produce antibody andantibody-like molecules by recombinant means and thereby generate genesequences that code for specific amino acid sequences found in thepolypeptide structure of the antibodies. Such antibodies can be producedby either cloning the gene sequences encoding the polypeptide chains ofsaid antibodies or by direct synthesis of said polypeptide chains, withassembly of the synthesized chains to form active tetrameric (H₂L₂)structures with affinity for specific epitopes and antigenicdeterminants. This has permitted the ready production of antibodieshaving sequences characteristic of neutralizing antibodies fromdifferent species and sources.

Regardless of the source of the antibodies, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, large cell cultures of laboratory orcommercial size, using transgenic plants, or by direct chemicalsynthesis employing no living organisms at any stage of the process, allantibodies have a similar overall 3 dimensional structure. Thisstructure is often given as H₂L₂ and refers to the fact that antibodiescommonly comprise two light (L) amino acid chains and 2 heavy (H) aminoacid chains. Both chains have regions capable of interacting with astructurally complementary antigenic target. The regions interactingwith the target are referred to as “variable” or “V” regions and arecharacterized by differences in amino acid sequence from antibodies ofdifferent antigenic specificity. The variable regions of either H or Lchains contain the amino acid sequences capable of specifically bindingto antigenic targets.

As used herein, the term “antigen binding region” refers to that portionof an antibody molecule which contains the amino acid residues thatinteract with an antigen and confer on the antibody its specificity andaffinity for the antigen. The antibody binding region includes the“framework” amino acid residues necessary to maintain the properconformation of the antigen-binding residues.

Within the variable regions of the H or L chains that provide for theantigen binding regions are smaller sequences dubbed “hypervariable”because of their extreme variability between antibodies of differingspecificity. Such hypervariable regions are also referred to as“complementarity determining regions” or “CDR” regions. These CDRregions account for the basic specificity of the antibody for aparticular antigenic determinant structure.

The CDRs represent non-contiguous stretches of amino acids within thevariable regions but, regardless of species, the positional locations ofthese critical amino acid sequences within the variable heavy and lightchain regions have been found to have similar locations within the aminoacid sequences of the variable chains. The variable heavy and lightchains of all antibodies each have three CDR regions, eachnon-contiguous with the others.

In all mammalian species, antibody peptides contain constant (i.e.,highly conserved) and variable regions, and, within the latter, thereare the CDRs and the so-called “framework regions” made up of amino acidsequences within the variable region of the heavy or light chain butoutside the CDRs.

Regarding the antigenic determinate recognized by the CDR regions of theantibody, this is also referred to as the “epitope.” In other words,epitope refers to that portion of any molecule capable of beingrecognized by, and bound by, an antibody (the corresponding antibodybinding region may be referred to as a paratope).

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce an antibody capable of binding to an epitope of that antigen.An antigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The term “antibody” is meant to include both intact immunoglobulinmolecules as well as portions, fragments, peptides and derivativesthereof such as, for example, Fab, Fab′, F(ab′)2, Fv, Fse, CDR regions,paratopes, or any portion or peptide sequence of the antibody that iscapable of binding an antigen or epitope. An antibody is said to be“capable of binding” a molecule if it is capable of specificallyreacting with the molecule to thereby bind the molecule to the antibody.

Antibody also includes chimeric antibodies, heterochimeric antibodies,caninized antibodies, or felinized antibodies, as well as fragments,portions, regions, peptides or derivatives thereof, provided by anyknown technique, such as, but not limited to, enzymatic cleavage,peptide synthesis, or recombinant techniques. Such antibodies of thepresent invention are capable of specifically binding at least one ofcanine IL-31 or feline IL-31. Antibody fragments or portions may lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding than anintact antibody. Examples of antibody fragments may be produced fromintact antibodies using methods well known in the art, for example byproteolytic cleavage with enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′).2 fragments). See, e.g., Wahl etal., 24 J. Nucl. Med. 316-25 (1983). Portions of antibodies may be madeby any of the above methods, or may be made by expressing a portion ofthe recombinant molecule. For example, the CDR region(s) of arecombinant antibody may be isolated and subcloned into the appropriateexpression vector. See, e.g., U.S. Pat. No. 6,680,053.

Clones 11 E1 2, 34D03 and 19D07 Nucleotide and Amino Acid Sequences

In some embodiments, the present invention provides for novel monoclonalantibodies that specifically bind to at least one of canine IL-31 orfeline IL-31. In one embodiment, a monoclonal antibody of the inventionbinds to canine IL-31 or feline IL-31 and prevents its binding to, andactivation of, its co-receptor complex comprising IL-31 receptor A(IL-31Ra) and Oncostatin-M-specific receptor (OsmR or IL-31Rb). Themonoclonal antibodies of the present invention are identified herein as“11E12”, “34D03” and “19D07”, which refers to the number assigned to itshybridoma clone. Herein, “11E12”, “34D03”, or “19D07” also refers to theportion of the monoclonal antibody, the paratope or CDRs, that bindspecifically with an IL-31 epitope identified as 11E12, 34D03, or 19D07because of its ability to bind the 11E1 2, 34D03, or 19D07 antibodies,respectively. The several recombinant, chimeric, heterochimeric,caninized and/or felinized forms of 11E1 2, 34D03 and 19D07 describedherein may be referred to by the same name.

In one embodiment, the present invention provides an isolated antibodyor antigen-binding portion thereof including at least one of thefollowing:

-   -   a variable heavy (VH) chain complementary determining region        (CDR)1 having theamino acid sequence YYDIN (SEQ ID NO: 1;        11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1), or NYGMS        (SEQ ID NO: 3; 34D03-VH-CDR1);    -   a variable heavy chain CDR2 having the amino acid sequence        WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2),        TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2), or        TISYGGSYTYYPDNIKG (SEQ ID NO: 6; 34D03-VH-CDR2);    -   a variable heavy chain CDR3 having the amino acid sequence        ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWVVGLAY (SEQ        ID NO: 8; 19D07-VH-CDR3), or VRGYGYDTMDY (SEQ ID NO: 9;        34D03-VH-CDR3); and    -   a variant thereof having one or more conservative amino acid        substitutions in at least one of CDR1, CDR2, or CDR3.

In another embodiment, the invention provides an isolated antibody orantigen-binding portion thereof including at least one of the followinggroup:

-   -   a variable light (V_(L)) chain comprising a complementary        determining region (CDR) 1 having the amino acid sequence        RASESVDNYGISFMH (SEQ ID NO: 10; 11E12-VL-CDR1),        KSSQSLLNSGNQKNYLA (SEQ ID NO: 11; 19D07-VL-CDR1), or        KASQSVSFAGTGLMH (SEQ ID NO: 12; 34D03-VL-CDR1);    -   a variable light chain CDR2 having the amino acid sequence        RASNLES (SEQ ID NO: 13; 11E12-VL-CDR2), GASTRES (SEQ ID NO: 14;        19D07-VL-CDR2), or RASNLEA (SEQ ID NO: 15; 34D03-VL-CDR2);    -   a variable light chain CDR3 having the amino acid sequence        QQSNKDPLT (SEQ ID NO: 16; 11E12-VL-CDR3), QNDYSYPYT (SEQ ID NO:        17; 19D07-VL-CDR3), or QQSREYPWT (SEQ ID NO: 18; 34D03-VL-CDR3);        and    -   a variant thereof having one or more conservative amino acid        substitutions in at least one of CDR1, CDR2, or CDR3.

In still other embodiments, an antibody having at least one of thevariable light chain CDRs described above, can further include at leastone of the following variable heavy chain CDRs:

-   -   a variable heavy chain complementary determining region (CDR)1        having the amino acid sequence YYDIN (SEQ ID NO: 1;        11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1), or NYGMS        (SEQ ID NO: 3; 34D03-VH-CDR1);    -   a variable heavy chain CDR2 having the amino acid sequence        WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2),        TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2), or        TISYGGSYTYYPDNIKG (SEQ ID NO: 6; 34D03-VH-CDR2);    -   a variable heavy chain CDR3 having the amino acid sequence        ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWVVGLAY (SEQ        ID NO: 8; 19D07-VH-CDR3), or VRGYGYDTMDY (SEQ ID NO: 9;        34D03-VH-CDR3); and    -   a variant thereof having one or more conservative amino acid        substitutions in at least one of CDR1, CDR2, or CDR3.

In some embodiments, the antibody can include at least one of thefollowing:

-   -   a) a variable light chain comprising

MU-11E12-VL) (SEQ ID NO: 19DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVETDDVATYYCQQ SNKDPLTFGAGTKLELK;,CAN-11E12-VL-cUn-FW2) (SEQ ID NO: 20DIVMTQTPLSLSVSPGEPASISCRASESVDNYGISFMHWYQQKPGQPPKLLIYRASNLESGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCQQ SNKDPLTFGAGTKLEIK;,CAN-11E12-VL-cUn-13) (SEQ ID NO: 21DIVMTQTPLSLSVSPGEPASISCRASESVDNYGISFMHWFQQKPGQSPQLLIYRASNLESGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCQQ SNKDPLTFGAGTKLEIK;,MU-19D07-VL) (SEQ ID NO: 22DIVMSQSPSSLSVSAGDKVTMSCKSSQSLLNSGNQKNYLAWYQQKPWQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDYSYPYTFGGGTKLEIK;,CAN-19D07-VL-998-1) (SEQ ID NO: 23EIVMTQSPASLSLSQEEKVTITCKSSQSLLNSGNQKNYLAWYQQKPGQAPKLLIYGASTRESGVPSRFSGSGSGTDFTSFTISSLEPEDVAVYY CQNDYSYPYTFGQGTKLEIK;,MU-34D03-VL) (SEQ ID NO: 24DILLTQSPASLAVSLGQRAIISCKASQSVSFAGTGLMHWYQQKPGQQPKLLIYRASNLEAGVPTRFSGSGSRTDFTLNIHPVEEEDAATYFCQQ SREYPWTFGGGTKLEIK;, orCAN-34D03-VL-998-1) (SEQ ID NO: 25EIVMTQSPASLSLSQEEKVTITCKASQSVSFAGTGLMHWYQQKPGQAPKLLIYRASNLEAGVPSRFSGSGSGTDFSFTISSLEPEDVAVYYCQQ SREYPWTFGQGTKLEIK;;

-   -   b) a variable heavy chain comprising

MU-11E12-VH) (SEQ ID NO: 26QVQLQQSGAELVKPGASVKLSCKASGYTFKYYDINWVRQRPEQGLEWIGWIFPGDGGTKYNETFKGKATLTTDKSSSTAYMQLSRLTSEDSAVYFCARGGTSVIRDAMDYWGQGTSVTVSS;, CAN-11E12-VH-415-1) (SEQ ID NO: 27EVQLVQSGAEVKKPGASVKVSCKTSGYTFKYYDINWVRQAPGAGLDWMGWIFPGDGGTKYNETFKGRVTLTADTSTSTAYMELSSLRAGDIAVYYCARGGTSVIRDAMDYWGQGTLVTVSS;, MU-19D07-VH) (SEQ ID NO: 28EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQIPEKRLEWVATITSGGGYTYSADSVKGRFTISRDNARNTLYLQMSSLRSEDTAVYYCARQNWVVGLAYWGQGTLVTVSA;, CAN-19D07-VH-400-1) (SEQ ID NO: 29EVQLVESGGDLVKPGGSLRLSCVASGFTFSSYDMSWVRQAPGKGLQWVATITSGGGYTYSADSVKGRFTISRDNARNTLYLQMNSLRSEDTAVYYCARQNWVVGLAYWGQGTLVTVSS;, MU-34D03-VH) (SEQ ID NO: 30EVQLVESGGDLVKPGGSLKLSCAASGFSFSNYGMSWVRQTPDKRLEWVATISYGGSYTYYPDNIKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCVRGYGYDTMDYWGQGTSVTVSS;, or CAN-34D03-VH-568-1) (SEQ ID NO: 31EVQLVESGGDLVKPGGSLRLSCVASGFTFSNYGMSWVRQAPGKGLQWVATISYGGSYTYYPDNIKGRFTISRDNAKNTLYLQMNSLRAEDTAMYYCVRGYGYDTMDYWGQGTLVTVSS;;and

-   -   c) variants thereof having one or more conservative amino acid        substitutions.

In other embodiments, the invention provides a host cell that producesan antibody described above.

The present invention also includes, within its scope, nucleotidesequences encoding the variable regions of the light and heavy chains ofthe anti-IL-31 antibody of the present invention. Included also withinthe scope of the invention is any nucleotide sequence that encodes theamino acid sequence of 11E12, 34D03 or 19D07 or peptides thereof.

In some embodiments, the invention provides an isolated nucleic acidincluding a nucleic acid sequence encoding at least one of thefollowing:

-   -   a variable heavy (V_(H)) chain complementary determining region        (CDR)1 having the amino acid sequence YYDIN (SEQ ID NO: 1;        11E12-VH-CDR1), SYDMS (SEQ ID NO: 2; 19D07-VH-CDR1), or NYGMS        (SEQ ID NO: 3; 34D03-VH-CDR1);    -   a variable heavy chain CDR2 having the amino acid sequence        WIFPGDGGTKYNETFKG (SEQ ID NO: 4; 11E12-VH-CDR2),        TITSGGGYTYSADSVKG (SEQ ID NO: 5; 19D07-VH-CDR2), or        TISYGGSYTYYPDNIKG (SEQ ID NO: 6; 34D03-VH-CDR2);    -   a variable heavy chain CDR3 having the amino acid sequence        ARGGTSVIRDAMDY (SEQ ID NO: 7; 11E12-VH-CDR3), ARQNWVVGLAY (SEQ        ID NO: 8; 19D07-VH-CDR3), or VRGYGYDTMDY (SEQ ID NO: 9;        34D03-VH-CDR3); and    -   a variant thereof having one or more conservative amino acid        substitutions in at least one of CDR1, CDR2, or CDR3.

In further embodiments, the isolated nucleic acid described above mayfurther include a nucleic acid sequence encoding at least one of thefollowing:

-   -   a variable light (V_(L)) chain comprising a complementary        determining (CDR) 1 having the amino acid sequence        RASESVDNYGISFMH (SEQ ID NO: 10; 11E12-VL-CDR1),        KSSQSLLNSGNQKNYLA (SEQ ID NO: 11; 19D07-VL-CDR1), or        KASQSVSFAGTGLMH (SEQ ID NO: 12; 34D03-VL-CDR1);    -   a variable light chain CDR2 having the amino acid sequence        RASNLES (SEQ ID NO: 13; 11E12-VL-CDR2), GASTRES (SEQ ID NO: 14;        19D07-VL-CDR2), or RASNLEA (SEQ ID NO: 15; 34D03-VL-CDR2);    -   a variable light chain CDR3 having the amino acid sequence        QQSNKDPLT (SEQ ID NO: 16; 11E12-VL-CDR3), QNDYSYPYT (SEQ ID NO:        17; 19D07-VL-CDR3), or QQSREYPWT (SEQ ID NO: 18; 34D03-VL-CDR3);        and    -   a variant thereof having one or more conservative amino acid        substitutions in at least one of CDR1, CDR2, or CDR3.

The present invention further provides a vector including at least oneof the nucleic acids described above.

Because the genetic code is degenerate, more than one codon can be usedto encode a particular amino acid. Using the genetic code, one or moredifferent nucleotide sequences can be identified, each of which would becapable of encoding the amino acid. The probability that a particularoligonucleotide will, in fact, constitute the actual XXX-encodingsequence can be estimated by considering abnormal base pairingrelationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic orprokaryotic cells expressing an anti-IL-31 antibody or portion. Such“codon usage rules” are disclosed by Lathe, et al., 183 J. Molec. Biol.1-12 (1985). Using the “codon usage rules” of Lathe, a single nucleotidesequence, or a set of nucleotide sequences, that contains a theoretical“most probable” nucleotide sequence capable of encoding anti-IL-31sequences can be identified. It is also intended that the antibodycoding regions for use in the present invention could also be providedby altering existing antibody genes using standard molecular biologicaltechniques that result in variants (agonists) of the antibodies andpeptides described herein. Such variants include, but are not limited todeletions, additions and substitutions in the amino acid sequence of theanti-IL-31 antibodies or peptides.

For example, one class of substitutions is conservative amino acidsubstitutions. Such substitutions are those that substitute a givenamino acid in an anti-IL-31antibody peptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Arg,replacements among the aromatic residues Phe, Tyr, and the like.Guidance concerning which amino acid changes are likely to bephenotypically silent is found in Bowie et al., 247 Science 1306-10(1990).

Variant or agonist anti-IL-31 antibodies or peptides may be fullyfunctional or may lack function in one or more activities. Fullyfunctional variants typically contain only conservative variations orvariations in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, or truncation or a substitution, insertion,inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as epitope binding or in vitro ADCC activity. Sites thatare critical for ligand-receptor binding can also be determined bystructural analysis such as crystallography, nuclear magnetic resonance,or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904(1992); de Vos et al., 255 Science 306-12 (1992).

Moreover, polypeptides often contain amino acids other than the twenty“naturally occurring” amino acids. Further, many amino acids, includingthe terminal amino acids, may be modified by natural processes, such asprocessing and other post-translational modifications, or by chemicalmodification techniques well known in the art. Known modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent crosslinks, formation of cystine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins-Structure and Molecular Properties (2nd ed., T. E.Creighton, W.H. Freeman & Co., NY, 1993). Many detailed reviews areavailable on this subject, such as by Wold, Posttranslational CovalentModification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983);Seifter et al. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663Ann. NY Acad. Sci. 48-62 (1992).

Accordingly, the antibodies and peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code.

Similarly, the additions and substitutions in the amino acid sequence aswell as variations, and modifications just described may be equallyapplicable to the amino acid sequence of the IL-31 antigen and/orepitope or peptides thereof, and are thus encompassed by the presentinvention. As mentioned above, the genes encoding a monoclonal antibodyaccording to the present invention is specifically effective in therecognition of IL-31.

Antibody Derivatives

Included within the scope of this invention are antibody derivatives. A“derivative” of an antibody contains additional chemical moieties notnormally a part of the protein. Covalent modifications of the proteinare included within the scope of this invention. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the antibody with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. For example,derivatization with bifunctional agents, well-known in the art, isuseful for cross-linking the antibody or fragment to a water-insolublesupport matrix or to other macromolecular carriers.

Derivatives also include radioactively labeled monoclonal antibodiesthat are labeled. For example, with radioactive iodine (¹²⁵I,¹¹³I)carbon (¹⁴C), sulfur (³⁵S), indium (¹¹¹In), tritium (³H) or thelike; conjugates of monoclonal antibodies with biotin or avidin, withenzymes, such as horseradish peroxidase, alkaline phosphatase,beta-D-galactosidase, glucose oxidase, glucoamylase, carboxylic acidanhydrase, acetylcholine esterase, lysozyme, malate dehydrogenase orglucose 6-phosphate dehydrogenase; and also conjugates of monoclonalantibodies with bioluminescent agents (such as luciferase),chemoluminescent agents (such as acridine esters) or fluorescent agents(such as phycobiliproteins).

Another derivative bifunctional antibody of the present invention is abispecific antibody, generated by combining parts of two separateantibodies that recognize two different antigenic groups. This may beachieved by crosslinking or recombinant techniques. Additionally,moieties may be added to the antibody or a portion thereof to increasehalf-life in vivo (e.g., by lengthening the time to clearance from theblood stream. Such techniques include, for example, adding PEG moieties(also termed pegilation), and are well-known in the art. See U.S.Patent. Appl. Pub. No. 20030031671.

Recombinant Expression of Antibodies

In some embodiments, the nucleic acids encoding a subject monoclonalantibody are introduced directly into a host cell, and the cell isincubated under conditions sufficient to induce expression of theencoded antibody. After the subject nucleic acids have been introducedinto a cell, the cell is typically incubated, normally at 37° C.,sometimes under selection, for a period of about 1-24 hours in order toallow for the expression of the antibody. In one embodiment, theantibody is secreted into the supernatant of the media in which the cellis growing.

Traditionally, monoclonal antibodies have been produced as nativemolecules in murine hybridoma lines. In addition to that technology, thepresent invention provides for recombinant DNA expression of monoclonalantibodies. This allows the production of caninized and felinizedantibodies, as well as a spectrum of antibody derivatives and fusionproteins in a host species of choice.

A nucleic acid sequence encoding at least one anti-IL-31 antibody,portion or polypeptide of the present invention may be recombined withvector DNA in accordance with conventional techniques, includingblunt-ended or staggered-ended termini for ligation, restriction enzymedigestion to provide appropriate termini, filling in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and ligation with appropriate ligases. Techniques for suchmanipulations are disclosed, e.g., by Maniatis et al., MOLECULARCLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and1989), and Ausubel et al. 1993 supra, may be used to construct nucleicacid sequences which encode a monoclonal antibody molecule or antigenbinding region thereof.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as anti-IL-31peptides or antibody portions in recoverable amounts. The precise natureof the regulatory regions needed for gene expression may vary fromorganism to organism, as is well known in the analogous art. See, e.g.,Sambrook et al., 2001 supra; Ausubel et al., 1993 supra.

The present invention accordingly encompasses the expression of ananti-IL-31 antibody or peptide, in either prokaryotic or eukaryoticcells. Suitable hosts include bacterial or eukaryotic hosts includingbacteria, yeast, insects, fungi, bird and mammalian cells either invivo, or in situ, or host cells of mammalian, insect, bird or yeastorigin. The mammalian cell or tissue may be of human, primate, hamster,rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but anyother mammalian cell may be used.

In one embodiment, the introduced nucleotide sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. See, e.g., Ausubel et al., 1993 supra.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Example prokaryotic vectors known in the art include plasmids such asthose capable of replication in E. coli (such as, for example, pBR322,ColE1, pSC101, pACYC 184, .pi.VX). Such plasmids are, for example,disclosed by Maniatis et al., 1989 supra; Ausubel et al, 1993 supra.Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids aredisclosed by Gryczan, in THE MOLEC. BIO. OF THE BACILLI 307-329(Academic Press, NY, 1982). Suitable Streptomyces plasmids includepIJ101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987)), andStreptomyces bacteriophages such as .phi.C31 (Chater et al., in SIXTHINT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido,Budapest, Hungary 1986). Pseudomonas plasmids are reviewed in John etal., 8 Rev. Infect. Dis. 693-704 (1986); Izaki, 33 Jpn. J. Bacteriol.729-42 (1978); and Ausubel et al., 1993 supra.

Alternatively, gene expression elements useful for the expression ofcDNA encoding anti-IL-31antibodies or peptides include, but are notlimited to (a) viral transcription promoters and their enhancerelements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell.Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl.Acad. Sci., USA 6777 (1982)), and Moloney murine leukemia virus LTR(Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions andpolyadenylation sites such as those derived from the SV40 late region(Okayarea et al., 1983), and (c) polyadenylation sites such as in SV40(Okayama et al., 1983).

Immunoglobulin cDNA genes can be expressed as described by Weidle etal., 51 Gene 21 (1987), using as expression elements the SV40 earlypromoter and its enhancer, the mouse immunoglobulin H chain promoterenhancers, SV40 late region mRNA splicing, rabbit S-globin interveningsequence, immunoglobulin and rabbit S-globin polyadenylation sites, andSV40 polyadenylation elements.

For immunoglobulin genes comprised of part cDNA, part genomic DNA(Whittle et al., 1 Protein Engin. 499 (1987)), the transcriptionalpromoter can be human cytomegalovirus, the promoter enhancers can becytomegalovirus and mouse/human immunoglobulin, and mRNA splicing andpolyadenylation regions can be the native chromosomal immunoglobulinsequences.

In one embodiment, for expression of cDNA genes in rodent cells, thetranscriptional promoter is a viral LTR sequence, the transcriptionalpromoter enhancers are either or both the mouse immunoglobulin heavychain enhancer and the viral LTR enhancer, the splice region contains anintron of greater than 31 bp, and the polyadenylation and transcriptiontermination regions are derived from the native chromosomal sequencecorresponding to the immunoglobulin chain being synthesized. In otherembodiments, cDNA sequences encoding other proteins are combined withthe above-recited expression elements to achieve expression of theproteins in mammalian cells.

Each fused gene can be assembled in, or inserted into, an expressionvector. Recipient cells capable of expressing the chimericimmunoglobulin chain gene product are then transfected singly with ananti-IL-31 peptide or chimeric H or chimeric L chain-encoding gene, orare co-transfected with a chimeric H and a chimeric L chain gene. Thetransfected recipient cells are cultured under conditions that permitexpression of the incorporated genes and the expressed immunoglobulinchains or intact antibodies or fragments are recovered from the culture.

In one embodiment, the fused genes encoding the anti-IL-31 peptide orchimeric H and L chains, or portions thereof are assembled in separateexpression vectors that are then used to co-transfect a recipient cell.Alternatively the fused genes encoding the chimeric H and L chains canbe assembled on the same expression vector.

For transfection of the expression vectors and production of thechimeric antibody, the recipient cell line may be a myeloma cell.Myeloma cells can synthesize, assemble and secrete immunoglobulinsencoded by transfected immunoglobulin genes and possess the mechanismfor glycosylation of the immunoglobulin. Myeloma cells can be grown inculture or in the peritoneal cavity of a mouse, where secretedimmunoglobulin can be obtained from ascites fluid. Other suitablerecipient cells include lymphoid cells such as B lymphocytes of human ornon-human origin, hybridoma cells of human or non-human origin, orinterspecies heterohybridoma cells.

The expression vector carrying a chimeric, caninized or felinizedantibody construct or anti-IL-31 polypeptide of the present inventioncan be introduced into an appropriate host cell by any of a variety ofsuitable means, including such biochemical means as transformation,transfection, conjugation, protoplast fusion, calciumphosphate-precipitation, and application with polycations such asdiethylaminoethyl (DEAE) dextran, and such mechanical means aselectroporation, direct microinjection, and microprojectile bombardment.Johnston et al., 240 Science 1538 (1988).

Yeast can provide substantial advantages over bacteria for theproduction of immunoglobulin H and L chains. Yeasts carry outpost-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies now exist which utilize strongpromoter sequences and high copy number plasmids which can be used forproduction of the desired proteins in yeast. Yeast recognizes leadersequences of cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e., pre-peptides). Hitzman et al., 11thInt'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France,1982).

Yeast gene expression systems can be routinely evaluated for the levelsof production, secretion and the stability of anti-IL-31 peptides,antibody and assembled murine and chimeric, heterochimeric, caninized,or felinized antibodies, fragments and regions thereof. Any of a seriesof yeast gene expression systems incorporating promoter and terminationelements from the actively expressed genes coding for glycolytic enzymesproduced in large quantities when yeasts are grown in media rich inglucose can be utilized. Known glycolytic genes can also provide veryefficient transcription control signals. For example, the promoter andterminator signals of the phosphoglycerate kinase (PGK) gene can beutilized. A number of approaches can be taken for evaluating optimalexpression plasmids for the expression of cloned immunoglobulin cDNAs inyeast. See Vol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford,UK 1985).

Bacterial strains can also be utilized as hosts for the production ofantibody molecules or peptides described by this invention. Plasmidvectors containing replicon and control sequences which are derived fromspecies compatible with a host cell are used in connection with thesebacterial hosts. The vector carries a replication site, as well asspecific genes which are capable of providing phenotypic selection intransformed cells. A number of approaches can be taken for evaluatingthe expression plasmids for the production of murine, chimeric,heterochimeric, caninized or felinized antibodies, fragments and regionsor antibody chains encoded by the cloned immunoglobulin cDNAs inbacteria (see Glover, 1985 supra; Ausubel, 1993 supra; Sambrook, 2001supra; Colligan et al., eds. Current Protocols in Immunology, John Wiley& Sons, NY, N.Y. (1994-2001); Colligan et al., eds. Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y. (1997-2001).

Host mammalian cells may be grown in vitro or in vivo. Mammalian cellsprovide post-translational modifications to immunoglobulin proteinmolecules including leader peptide removal, folding and assembly of Hand L chains, glycosylation of the antibody molecules, and secretion offunctional antibody protein.

Mammalian cells which can be useful as hosts for the production ofantibody proteins, in addition to the cells of lymphoid origin describedabove, include cells of fibroblast origin, such as Vero (ATCC CRL 81) orCHO-K1 (ATCC CRL 61) cells.

Many vector systems are available for the expression of clonedanti-IL-31 peptides H and L chain genes in mammalian cells (see Glover,1985 supra). Different approaches can be followed to obtain completeH₂L₂ antibodies. It is possible to co-express H and L chains in the samecells to achieve intracellular association and linkage of H and L chainsinto complete tetrameric H₂L₂ antibodies and/or anti-IL-31 peptides. Theco-expression can occur by using either the same or different plasmidsin the same host. Genes for both H and L chains and/or anti-IL-31peptides can be placed into the same plasmid, which is then transfectedinto cells, thereby selecting directly for cells that express bothchains. Alternatively, cells can be transfected first with a plasmidencoding one chain, for example the L chain, followed by transfection ofthe resulting cell line with an H chain plasmid containing a secondselectable marker. Cell lines producing anti-IL-31 peptides and/or H₂L₂molecules via either route could be transfected with plasmids encodingadditional copies of peptides, H, L, or H plus L chains in conjunctionwith additional selectable markers to generate cell lines with enhancedproperties, such as higher production of assembled H₂L₂ antibodymolecules or enhanced stability of the transfected cell lines.

For long-term, high-yield production of recombinant antibodies, stableexpression may be used. For example, cell lines, which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with immunoglobulin expression cassettes and a selectablemarker. Following the introduction of the foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and grow to form foci which inturn can be cloned and expanded into cell lines. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds/components that interact directly or indirectly with theantibody molecule.

Once an antibody of the invention has been produced, it may be purifiedby any method known in the art for purification of an immunoglobulinmolecule, for example, by chromatography (e.g., ion exchange, affinity,particularly affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins. Inmany embodiments, antibodies are secreted from the cell into culturemedium and harvested from the culture medium.

Pharmaceutical Applications

The anti-IL-31 antibodies or peptides of the present invention can beused for example in the treatment of pruritic and/or allergic conditionsin companion animals, such as dogs and cats. More specifically, theinvention further provides for a pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and, as activeingredient, an antibody or peptide according to the invention. Theantibody can be a chimeric, heterochimeric, caninized, or felinizedantibody according to the present invention. Intact immunoglobulins ortheir binding fragments, such as Fab, are also envisioned. Theantibodyand pharmaceutical compositions thereof of this invention areuseful for parenteral administration, e.g., subcutaneously,intramuscularly or intravenously.

Anti-IL-31 antibodies and/or peptides of the present invention can beadministered either as individual therapeutic agents or in combinationwith other therapeutic agents. They can be administered alone, but aregenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

Administration of the antibodies disclosed herein may be carried out byany suitable means, including parenteral injection (such asintraperitoneal, subcutaneous, or intramuscular injection), orally, orby topical administration of the antibodies (typically carried in apharmaceutical formulation) to an airway surface. Topical administrationto an airway surface can be carried out by intranasal administration(e.g., by use of dropper, swab, or inhaler). Topical administration ofthe antibodies to an airway surface can also be carried out byinhalation administration, such as by creating respirable particles of apharmaceutical formulation (including both solid and liquid particles)containing the antibodies as an aerosol suspension, and then causing thesubject to inhale the respirable particles. Methods and apparatus foradministering respirable particles of pharmaceutical formulations arewell known, and any conventional technique can be employed. Oraladministration may be, for example, in the form of an ingestable liquidor solid formulation.

In some desired embodiments, the antibodies are administered byparenteral injection. For parenteral administration, anti-IL-31antibodies or peptides can be formulated as a solution, suspension,emulsion or lyophilized powder in association with a pharmaceuticallyacceptable parenteral vehicle. For example the vehicle may be a solutionof the antibody or a cocktail thereof dissolved in an acceptablecarrier, such as an aqueous carrier such vehicles are water, saline,Ringer's solution, dextrose solution, trehalose or sucrose solution, or5% serum albumin, 0.4% saline, 0.3% glycine and the like. Liposomes andnonaqueous vehicles such as fixed oils can also be used. These solutionsare sterile and generally free of particulate matter. These compositionsmay be sterilized by conventional, well known sterilization techniques.The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjustment agents and thelike, for example sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate, etc. The concentration of antibody inthese formulations can vary widely, for example from less than about0.5%, usually at or at least about 1% to as much as 15% or 20% by weightand will be selected primarily based on fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.The vehicle or lyophilized powder can contain additives that maintainisotonicity (e.g., sodium chloride, mannitol) and chemical stability(e.g., buffers and preservatives). The formulation is sterilized bycommonly used techniques.

Actual methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in, for example, REMINGTON'S PHARMA. SCI. (15th ed., MackPub. Co., Easton, Pa., 1980).

The antibodies of this invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immune globulins. Anysuitable lyophilization and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilizationand reconstitution can lead to varying degrees of antibody activity lossand that use levels may have to be adjusted to compensate.

The compositions containing the present antibodies or a cocktail thereofcan be administered for prevention of recurrence and/or therapeutictreatments for existing disease. Suitable pharmaceutical carriers aredescribed in the most recent edition of REMINGTON'S PHARMACEUTICALSCIENCES, a standard reference text in this field of art.

In therapeutic application, compositions are administered to a subjectalready suffering from a disease, in an amount sufficient to cure or atleast partially arrest or alleviate the disease and its complications.An amount adequate to accomplish this is defined as a “therapeuticallyeffective dose” or a “therapeutically effective amount”. Amountseffective for this use will depend upon the severity of the disease andthe general state of the subject's own immune system, but generallyrange from about 0.1 mg antibody per kg body weight to about 10 mgantibody per kg body weight, preferably about 0.3 mg antibody per kg ofbody weight to about 5 mg of antibody per kg of body weight. In view ofthe minimization of extraneous substances and the lower probability of“foreign substance” rejections which are achieved by the presentcanine-like and feline-like antibodies of this invention, it may bepossible to administer substantial excesses of these antibodies.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms kind of concurrenttreatment, frequency of treatment, and the effect desired.

As a non-limiting example, treatment of IL-31-related pathologies indogs or cats can be provided as a biweekly or monthly dosage ofanti-IL-31 antibodies of the present invention in the dosage rangedescribed above.

Example antibodies for canine or feline therapeutic use are highaffinity (these may also be high avidity) antibodies, and fragments,regions and derivatives thereof having potent in vivo anti-IL-31activity, according to the present invention.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingveterinarian. In any event, the pharmaceutical formulations shouldprovide a quantity of the antibody(ies) of this invention sufficient toeffectively treat the subject.

Diagnostic Applications

The present invention also provides the above anti-IL-31 antibodies andpeptides for use in diagnostic methods for detecting IL-31 in companionanimals known to be or suspected of having a puritic and/or allergiccondition.

Anti-IL-31 antibodies and/or peptides of the present invention areuseful for immunoassays which detect or quantitate IL-31, or anti-IL-31antibodies, in a sample. An immunoassay for IL-31 typically comprisesincubating a clinical or biological sample in the presence of adetectably labeled high affinity (or high avidity) anti-IL-31 antibodyor polypeptide of the present invention capable of selectively bindingto IL-31, and detecting the labeled peptide or antibody which is boundin a sample. Various clinical assay procedures are well known in theart. See, e.g., IMMUNOASSAYS FOR THE 80'S (Voller et al., eds., Univ.Park, 1981). Such samples include tissue biopsy, blood, serum, and fecalsamples, or liquids collected from animal subjects and subjected toELISA analysis as described below.

In some embodiments, the binding of antigen to antibody is detectedwithout the use of a solid support. For example, the binding of antigento antibody can be detected in a liquid format.

In other embodiments, an anti-IL-31 antibody or polypeptide can, forexample, be fixed to nitrocellulose, or another solid support which iscapable of immobilizing cells, cell particles or soluble proteins. Thesupport can then be washed with suitable buffers followed by treatmentwith the detectably labeled IL-31-specific peptide or antibody. Thesolid phase support can then be washed with the buffer a second time toremove unbound peptide or antibody. The amount of bound label on thesolid support can then be detected by known method steps.

“Solid phase support” or “carrier” refers to any support capable ofbinding peptide, antigen, or antibody. Well-known supports or carriers,include glass, polystyrene, polypropylene, polyethylene,polyvinylidenefluoride (PVDF), dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble to some extent or insolublefor the purposes of the present invention. The support material can havevirtually any possible structural configuration so long as the coupledmolecule is capable of binding to IL-31 or an anti-IL-31 antibody. Thus,the support configuration can be spherical, as in a bead, orcylindrical, as in the inside surface of a test tube, or the externalsurface of a rod. Alternatively, the surface can be flat, such as asheet, culture dish, test strip, etc. For example, supports may includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody, peptide or antigen, or canascertain the same by routine experimentation.

Well known method steps can determine binding activity of a given lot ofanti-IL-31 peptide and/or antibody. Those skilled in the art candetermine operative and optimal assay conditions by routineexperimentation.

Detectably labeling an IL-31-specific peptide and/or antibody can beaccomplished by linking to an enzyme for use in an enzyme immunoassay(EIA), or enzyme-linked immunosorbent assay (ELISA). The linked enzymereacts with the exposed substrate to generate a chemical moiety whichcan be detected, for example, by spectrophotometric, fluorometric or byvisual means. Enzymes which can be used to detectably label theIL-31-specific antibodies of the present invention include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

By radioactively labeling the IL-31-specific antibodies, it is possibleto detect IL-31 through the use of a radioimmunoassay (RIA). See Work etal., LAB. TECHNIQUES & BIOCHEM. 1N MOLEC. Bio. (No. Holland Pub. Co.,NY, 1978). The radioactive isotope can be detected by such means as theuse of a gamma counter or a scintillation counter or by autoradiography.Isotopes which are particularly useful for the purpose of the presentinvention include: ³H, ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C, and ¹²⁵I.

It is also possible to label the IL-31-specific antibodies with afluorescent compound. When the fluorescent labeled antibody is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The IL-31-specific antibodies can also be delectably labeled usingfluorescence-emitting metals such a ¹²⁵Eu, or others of the lanthanideseries. These metals can be attached to the IL-31—specific antibodyusing such metal chelating groups as diethylenetriaminepentaacetic acid(DTPA) or ethylenediamine-tetraacetic acid (EDTA).

The IL-31-specific antibodies also can be detectably labeled by couplingto a chemiluminescent compound. The presence of the chemiluminescentlylabeled antibody is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound can be used to label theIL-31-specific antibody, portion, fragment, polypeptide, or derivativeof the present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Detection of the IL-31-specific antibody, portion, fragment,polypeptide, or derivative can be accomplished by a scintillationcounter, for example, if the detectable label is a radioactive gammaemitter, or by a fluorometer, for example, if the label is a fluorescentmaterial. In the case of an enzyme label, the detection can beaccomplished by colorometric methods which employ a substrate for theenzyme. Detection can also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

For the purposes of the present invention, the IL-31 which is detectedby the above assays can be present in a biological sample. Any samplecontaining IL-31 may be used. For example, the sample is a biologicalfluid such as, for example, blood, serum, lymph, urine, feces,inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissueextract or homogenate, and the like. The invention is not limited toassays using only these samples, however, it being possible for one ofordinary skill in the art, in light of the present specification, todetermine suitable conditions which allow the use of other samples.

In situ detection can be accomplished by removing a histologicalspecimen from an animal subject, and providing the combination oflabeled antibodies of the present invention to such a specimen. Theantibody (or portion thereof) may be provided by applying or byoverlaying the labeled antibody (or portion) to a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of IL-31 but also the distribution of IL-31 in theexamined tissue. Using the present invention, those of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

The antibody, fragment or derivative of the present invention can beadapted for utilization in an immunometric assay, also known as a“two-site” or “sandwich” assay. In a typical immunometric assay, aquantity of unlabeled antibody (or fragment of antibody) is bound to asolid support that is insoluble in the fluid being tested and a quantityof detectably labeled soluble antibody is added to permit detectionand/or quantification of the ternary complex formed between solid-phaseantibody, antigen, and labeled antibody.

The antibodies may be used to quantitatively or qualitatively detect theIL-31 in a sample or to detect presence of cells that express the IL-31.This can be accomplished by immunofluorescence techniques employing afluorescently labeled antibody (see below) coupled with fluorescencemicroscopy, flow cytometric, or fluorometric detection. For diagnosticpurposes, the antibodies may either be labeled or unlabeled. Unlabeledantibodies can be used in combination with other labeled antibodies(second antibodies) that are reactive with the antibody, such asantibodies specific for canine or feline immunoglobulin constantregions. Alternatively, the antibodies can be directly labeled. A widevariety of labels may be employed, such as radionuclides, fluors,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands(particularly haptens), etc. Numerous types of immunoassays, such asthose discussed previously are available and are well known to thoseskilled in the art.

In one embodiment, the diagnostic method for detecting IL-31 is alateral flow immunoassay test. This is also known as theimmunochromatographic assay, Rapid ImmunoMigration (RIM™) or strip test.Lateral flow immunoassays are essentially immunoassays adapted tooperate along a single axis to suit the test strip format. A number ofvariations of the technology have been developed into commercialproducts, but they all operate according to the same basic principle. Atypical test strip consists of the following components: (1) samplepad—an absorbent pad onto which the test sample is applied; (2)conjugate or reagent pad—this contains antibodies specific to the targetanalyte conjugated to colored particles (usually colloidal goldparticles, or latex microspheres); (3) reaction membrane—typically ahydrophobic nitrocellulose or cellulose acetate membrane onto whichanti-target analyte antibodies are immobilized in a line across themembrane as a capture zone or test line (a control zone may also bepresent, containing antibodies specific for the conjugate antibodies);and (4) wick or waste reservoir—a further absorbent pad designed to drawthe sample across the reaction membrane by capillary action and collectit. The components of the strip are usually fixed to an inert backingmaterial and may be presented in a simple dipstick format or within aplastic casing with a sample port and reaction window showing thecapture and control zones.

There are two main types of lateral flow immunoassay used inmicrobiological testing: double antibody sandwich assays and competitiveassays. In the double antibody sandwich format, the sample migrates fromthe sample pad through the conjugate pad where any target analytepresent will bind to the conjugate. The sample then continues to migrateacross the membrane until it reaches the capture zone where thetarget/conjugate complex will bind to the immobilized antibodiesproducing a visible line on the membrane. The sample then migratesfurther along the strip until it reaches the control zone, where excessconjugate will bind and produce a second visible line on the membrane.This control line indicates that the sample has migrated across themembrane as intended. Two clear lines on the membrane is a positiveresult. A single line in the control zone is a negative result.Competitive assays differ from the double antibody sandwich format inthat the conjugate pad contains antibodies that are already bound to thetarget analyte, or to an analogue of it. If the target analyte ispresent in the sample it will therefore not bind with the conjugate andwill remain unlabelled. As the sample migrates along the membrane andreaches the capture zone an excess of unlabelled analyte will bind tothe immobilized antibodies and block the capture of the conjugate, sothat no visible line is produced. The unbound conjugate will then bindto the antibodies in the control zone producing a visible control line.A single control line on the membrane is a positive result. Two visiblelines in the capture and control zones is a negative result. However, ifan excess of unlabelled target analyte is not present, a weak line maybe produced in the capture zone, indicating an inconclusive result.There are a number of variations on lateral flow technology. The capturezone on the membrane may contain immobilized antigens orenzymes—depending on the target analyte—rather than antibodies. It isalso possible to apply multiple capture zones to create a multiplextest. For example, commercial test strips able to detect both EHEC Shigatoxins ST1 and ST2 separately in the same sample have been developed.

Importantly, the antibodies of the present invention may be helpful indiagnosing a pruritic and/or allergic in dogs or cats. Morespecifically, the antibody of the present invention may identify theoverexpression of IL-31 in companion animals. Thus, the antibody of thepresent invention may provide an important immunohistochemistry tool.

The antibodies of the present invention may be used on antibody arrays,highly suitable for measuring gene expression profiles.

Kits

Also included within the scope of the present invention are kits forpracticing the subject methods. The kits at least include one or more ofthe antibodies of the present invention, a nucleic acid encoding thesame, or a cell containing the same. In one embodiment, an antibody ofthe present invention may be provided, usually in a lyophilized form, ina container. The antibodies, which may be conjugated to a label ortoxin, or unconjugated, are typically included in the kits with buffers,such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inertproteins, e.g., serum albumin, or the like. Generally, these materialswill be present in less than 5% wt. based on the amount of activeantibody, and usually present in total amount of at least about 0.001%wt. based again on the antibody concentration. Frequently, it will bedesirable to include an inert extender or excipient to dilute the activeingredients, where the excipient may be present in from about 1% to 99%wt. of the total composition. Where a second antibody capable of bindingto the primary antibody is employed in an assay, this will usually bepresent in a separate vial. The second antibody is typically conjugatedto a label and formulated in an analogous manner with the antibodyformulations described above. The kit will generally also include a setof instructions for use.

In one embodiment, a kit according to the present invention is a teststrip kit (lateral flow immunoassay kit) useful for detecting canine orfeline IL-31 protein in a sample. Such a test strip will typicallyinclude a sample pad onto which the test sample is applied; a conjugateor reagent pad containing an antibody specific to canine or felineIL-31, wherein the antibody is conjugated to colored particles (usuallycolloidal gold particles); a reaction membrane onto which anti-IL-31antibodies are immobilized in a line across the membrane as a capturezone or test line (a control zone may also be present, containingantibodies specific for the conjugate antibodies); and a furtherabsorbent pad designed to draw the sample across the reaction membraneby capillary action and collect it. The test strip kit will generallyalso include directions for use.

The invention will now be described further by the non-limiting examplesbelow.

EXAMPLES Example 1 Identification of Mouse Monoclonal AntibodiesRecognizing Canine Interleukin 31 (IL-31 )

Recombinant canine IL-31 was created in CHO cells using the CHROMOS ACE(Artificial Chromosome Expression) system (Chromos Molecular Systems,Inc., Burnaby, British Columbia) to generate the secreted canine IL-31protein having the sequence of SEQ ID NO: 32. This protein is encoded bythe nucleotide sequence of SEQ ID NO: 33. Conditioned medium from 400 mlof cell culture (CHO cell line) was obtained and dialyzed against 10volumes of QA buffer (20 mM Tris pH 8.0, 20 mM NaCl) for 4.5 hours.Dialyzed medium was 0.2 um filtered and loaded at 1 ml/min onto aSOURCE™ Q column (GE Healthcare, Uppsala, Sweden) pre-equilibrated withQA buffer. Protein was eluted using a multi step linear gradient. Themajority of IL-31 remained in the flow through (FT) fraction, a smallamount of IL-31 eluted early in the gradient. Identity of the proteinwas previously confirmed by Western immunoblotting, and Mass-Spectro(MS) analysis of a tryptic digest. Protein in the FT fraction wasconcentrated 4-5 fold and dialyzed overnight against Phosphate BufferedSaline (PBS) at 4° C. Stability of the protein was checked followingdialysis into PBS. No precipitation was observed, and no proteolysis wasobserved after several days at 4° C. De-glycosylation experiments usingN-glycosidase F resulted in the protein condensing down to a single bandof ˜15 kDa on SDS-PAGE. Protein concentration was determined using abicinchoninic assay (BCA assay) with Bovine Serum Albumin (BSA) as astandard (ThermoFisher Scientific, Inc., Rockford, Ill.). The proteinsolution was split into aliquots, snap frozen (liquid N₂) and stored at−80° C.

Mouse monoclonal antibodies were identified using standard immunizationsof female CF-1 mice with recombinant canine IL-31 produced in CHO cells.Titers from immunized mice were determined using an enzyme linkedimmunosorbent assay (ELISA). Canine IL-31 (50 ng/well) was immobilizedto polystyrene microplates and used as a capture antigen. Serum fromimmunized mice was diluted in phosphate buffered saline with 0.05%tween-20 (PBST). The presence of mouse anti-canine IL-31 antibodies wasdetected with a Horse Radish Peroxidase (HRP)-conjugated goat anti-mousesecondary antibody (Kirkegard & Perry Laboratories, Inc. (KPL, Inc.),Gaithersburg, Md.). Following addition of a chromogenic substrate(SureBlue Reserve TMB 1-Component Microwell Peroxidase Substrate, KPL,Inc., Gaithersburg, Md.) and a ten minute incubation at room temperature(RT) the reaction was stopped with the addition of 100 μL of 0.1 N HCl.The absorbance of each well was determined at an optical density (OD) of450 nm. FIG. 6 summarizes the antibody response of individual miceimmunized with canine IL-31. A pool of donor splenocytes from mice 3 and4 were used for fusion. Following fusion and screening for anti IL-31binding via direct ELISA, 100 wells were chosen for expansion andsecondary screening of anti IL-31 activity. Secondary screeningconfirmed that 81 fusions retained the ability to produce anti IL-31antibodies. Frozen cell stocks and supernatants from these 81 candidateswere preserved for further evaluation.

To identify candidates with inhibitory activity, all 81 supernatantswere assessed for their ability to affect IL-31-mediated pSTAT signalingin a cell-based assay. This cell-based assay measures pSTAT signaling incanine DH-82 monocyte cells pre-treated for 24 hours with canine gammainterferon (R&D Systems, Minneapolis, Minn.) at 10 ng/mL and serumstarved for 2 hours prior to IL-31 treatment to increase IL-31 receptorexpression. Following this pre-treatment, recombinant canine IL-31 isadded at 1 μg/mL for 5 minutes and STAT phosphorylation is evaluatedusing the Alpha Screen technology (Perkin Elmer. Waltham, Mass.). Sinceantibody concentrations and purity are unknown in hybridomasupernatants, these supernatants were qualitatively measured for theirability to inhibit STAT phosphorylation following a 1 hour co-incubationwith 1 μg/ml IL-31 using 1:2 or 1:20 dilutions of the supernatants. Thisexperiment identified 31 supernatants that inhibited >50% of the STATphosphorylation relative to untreated wells thereby justifyingpurification and further characterization.

Following purification and quantitation of each monoclonal antibody(mAb), the IC₅₀ values of all 31 antibodies were evaluated in the DH-82cell assay. Based on the resulting IC₅₀ values and competitive ELISAs todefine antibody classes based on epitope bins, three antibodiesdescribed in Table 1 were moved forward for further characterization,11E12, 19D07, and 34D03.

TABLE 1 Antibody HC isotype LC isotype 11E12 G1/2b kappa 19D07 2b kappa34D03 G1 kappa

Example 2 DNA Sequences Encoding 11E12, 19D07 and 34D03 Antibodies

Ribonucleic acid (RNA) was isolated from hybridoma cells 11E12, 19D07,and 34D03 using the Rneasy—mini kit (Qiagen, Inc., Germantown, Md.) asdescribed by the manufacturer. One million frozen cells from eachhybridoma were harvested by centrifugation and RNA was purified fromcell lysates using the Rneasy spin column according to method describedin the protocol. RNA was eluted from each column and used immediatelyfor quantitation and cDNA preparation. The RNA was analyzed for yieldand purity by measuring it's absorbance at 260 nm and 280 nm using aGeneQuant pro spectrophotometer (GE Healthcare, Uppsala, Sweden).Following isolation, the remaining RNA was stored at −80° C. for furtheruse.

Oligonucletide primers designed for amplification of the mouseimmunoglobulin (Ig) variable domains were used according to themanufacturer's instructions (EMD Chemicals, Inc., Gibbstown, N.J.). cDNAwas prepared from total hybridoma RNA by reverse transcription (RT)using the thermoscript RT kit (Invitrogen Corp., Carlsbad, Calif.)according to the manufacturer's instructions. 200-400 ng of RNA fromeach hybridoma was added to an individual reaction tube containing a 3′Ig constant region primer. The 3′ constant Ig primer is positionedproximal to the variable Ig region and will transcribe first strand cDNArepresenting the variable region of the mouse antibody. For eachhybridoma RNA, an individual RT reaction was performed using a 3′constant heavy chain and 3′ constant kappa light chain primer.

cDNA from each hybridoma were used as a template in a polymerase chainreaction (PCR) to amplify the variable IgG heavy and kappa light chaincDNA for the purpose of sequence determination. Multiple reactions wereperformed for each PCR using a degenerate 5′ primer or primer poolsdesigned to anneal to the signal sequence-coding regions of the mouse Igvariable domain. Separate PCR reactions were performed with a degenerateprimer or primer pools for amplification of murine variable heavy andvariable light chain regions (FIG. 7). PCR was performed with 1 μI ofthe cDNA reaction using the Expand High Fidelity DNA polymerase kit(Roche Diagnostics Corp., Indianapolis, Ind.) according to themanufacturers protocol. Thermocycling parameters for the PCR were asfollows; 94° C. for 2 min., 35 cycles (94° C. 15 sec., 55° C. 30 sec.,72° C. 1 min.), 72° C. 7 min. Fragments amplified from the PCR wereseparated by gel electrophoresis on a 1% agarose gel and purified usingQiagen gel extraction kit (Qiagen, Inc., Germantown, Md.). Forwardprimers for the heavy and light chain variable region incorporate EcoRIor Sall (New England Biolabs (NEB), Inc., Ipswich, Mass.) sites andreverse heavy and light chain variable, HindIII (NEB Inc., Ipswich,Mass.) to facilitate cloning into the pUC19 plasmid. Purified PCRfragments and pUC19 plasmid were digested with the above restrictionendonucleases at 37° C. for 1-2 hrs. Following digestion, PCR fragmentswere purified using a Qiaquick PCR cleanup kit (Qiagen, Inc.,Germantown, Md.). Digested plasmid was separated by gel electrophoresison a 1% agarose gel and purified using Qiagen gel extraction kit.Purified PCR fragments representing variable IgG heavy and kappa lightchain DNA were ligated into pUC19 plasmid using T4 DNA ligase andligation buffer (NEB, Inc., Ipswich, Mass.) at 4° C. overnight. 3 μl ofeach ligation reaction was used to transform E. coli TOP10 cells(Invitrogen Corp., Carlsbad, Calif.).

Plasmids were isolated from positive clones representing the variableregions of each hybridoma using a Qiagen mini prep kit (Qiagen 27106)according to the manufacturer's protocol. M13 forward and reverseprimers were used to amplify DNA sequence for each cloned insert usingthe Big Dye sequencing reaction (Applied Biosystems by Life TechnologiesCorp., Carlsbad, Calif.) according to manufacturer's protocol.Sequencing reactions were purified using a 96 well purification kit(Zymo Research, Irvine, Calif.) according to the manufacturer'sprotocol. Samples were loaded onto an ABI-3730 capillary sequencer andresulting sequence traces were analyzed using Sequencher (GeneCodes v.4.2) for presence of complete open reading frames. The murine anticanine IL-31 variable sequences determined for each antibody are asfollows, 11E12 variable light chain (Seq ID NO:19 MU-11E12-VL, thecorresponding nucleotide sequence for which is SEQ ID NO: 34), 11E12variable heavy chain (Seq ID NO: 26 MU-11E12-VH, the correspondingnucleotide sequence for which is SEQ ID NO: 35), 19D07 variable lightchain (Seq ID NO:22 MU-19D07-VL, the corresponding nucleotide sequencefor which is SEQ ID NO: 36), 19D07 variable heavy chain (Seq ID NO: 28MU-19D07-VH, the corresponding nucleotide sequence for which is SEQ IDNO: 37), 34D03 variable light chain (Seq ID NO:24 MU-34D03-VL, thecorresponding nucleotide sequence for which is SEQ ID NO: 38), and 34D03variable heavy chain (Seq ID NO:30 MU-34D03-VH, the correspondingnucleotide sequence for which is SEQ ID NO: 39).

To confirm the validity of cDNA sequence derived from each antibodiesvariable heavy and light chains, N-terminal sequence analysis wascarried out on purified mAb protein using Edman degradation on anApplied Biosystems model 494 gas phase protein sequencer. Table 2 belowdescribes the confirmation of variable light chain sequences forantibodies 11E1 2 and 34D03 and the variable heavy sequence of 34D03.The N-terminal amino acid of the variable heavy chain of antibody 11E12,derived by translation of the cDNA sequence, was determined to beglutamine. Glutamine, as amino terminal residue of a protein, canspontaneously undergoe cyclization to pyroglutamic acid preventingsequence determination by Edman degradation (Chelius et al., Anal Chem.2006 78(7):2370-6).

TABLE 2* Variable Light Chain Translated N-terminal AntibodycDNA Sequence Sequence 11E12 DIVLT DIVLT 19D07 DIVMS not tested 34D03DILLT DILLT Variable Heavy Chain Translated N-terminal AntibodycDNA Sequence Sequence 11E12 QVQLQ blocked 19D07 EVKLV not tested 34D03EVQLV EVQLV *In Table 2, “DIVLT” corresponds to residues 1-5 of SEQ IDNO: 19, “DIVMS” corresponds to residues 1-5 of SEQ ID NO: 22, “DILLT”corresponds to residues 1-5 of SEQ ID NO: 24, “QVQLQ” corresponds toresidues 1-5 of SEQ ID NO: 26, “EVKLV” corresponds to residues 1-5 ofSEQ ID NO: 28, and “EVQLV” corresponds to residues 1-5 of SEQ ID NO: 30.

Example 3 Construction of 11E12, 19D07 and 34D03 Chimeric Antibodies

As described above, antibodies are composed of a homodimer pairing oftwo heterodimeric proteins. Each protein chain (one heavy and one light)of the heterodimer consists of a variable domain and a constant domain.Each variable domain contains three complementary determining regions(CDRs) which contribute to antigen binding. CDRs are separated in thevariable domain by framework regions which provide a scaffold for properspatial presentation of the binding sites on the antibody. Together, theCDR and framework regions contribute to the antibodies ability to bindits cognate antigen (FIG. 2).

As further described above, a chimeric antibody consists of the variablesequence (both CDR and framework) from the mouse antibody (as determinedfrom the above sequence analysis) grafted onto the respective heavy andlight constant regions of a canine IgG molecule (FIG. 3). As thevariable domain is responsible for antigen binding, grafting of thefully mouse variable domain onto canine constant region is expected tohave little or no impact on the antibody's ability to bind the IL-31immunogen.

To simultaneously confirm that the correct sequence of the heavy andlight chain variable regions were identified and to produce recombinant,homogenous material, expression vectors to produce the chimericantibodies in mammalian expression systems were generated. Forward andreverse primers were designed to amplify the mouse heavy and light chainvariable region of antibody sequence derived from hybridomas 11E12,19D07, and 34D03. A unique restriction endonuclease site, Kozakconsensus sequence and, secretion leader sequence were incorporated intoeach forward primer to facilitate expression and secretion of therecombinant antibody from a mammalian cell line. Each reverse primer wasdesigned to amplify each respective variable heavy and light chain andincluded a unique restriction site to facilitate cloning. PCR wasperformed to amplify each heavy and light chain using cloned hybridomavariable chain antibody DNA as a template for each reaction. Each PCRproduct was cloned into a mammalian expression plasmid containing eitherthe canine IgG heavy (referred to herein as HC-64 or HC-65) or lightchain (referred to herein as kappa) constant regions based on sequencesfrom GenBank accession numbers AF354264 or AF354265 and XP_532962respectively. The amino acid and nucleotide sequences of HC-64 arerepresented by SEQ ID NOs: 40 and 41, respectively. The amino acid andnucleotide sequences of HC-65 are represented by SEQ ID NOs: 42 and 43,respectively. The amino acid and nucleotide sequences of kappa arerepresented by SEQ ID NOs: 44 and 45, respectively. The plasmidsencoding each heavy and light chain, under the control of the CMVpromoter, were co-transfected into HEK 293 cells using standardlipofectamine methods. Following six days of expression, chimeric mAbswere purified from 30 ml of transiently transfected HEK293FS cellsupernatants using MabSelect SuRe protein A resin (GE Healthcare,Uppsala, Sweden) according to standard methods for protein purification.Eluted fractions were pooled, concentrated to ˜500 ul using a 10,000nominal MW cutoff Nanosep Omega centrifugal device (Pall Corp., PortWashington, N.Y.), dialyzed overnight at 4° C. in 1× PBS, pH7.2 andstored at 4° C. for further use.

Expression of chimeric canine IgG was assessed using SDS polyacrylamideelectrophoresis (SDS PAGE) under native and denaturing conditions.Monoclonal antibodies (mAbs) from each transfection were separated on a4-12% Bis Tris gel using SDS MES running buffer according to themanufacturers protocol (Invitrogen Corp., Carlsbad, Calif.). Followingelectrophoresis, proteins were visualized with Simply Blue CoomassieStain (Invitrogen Corp., Carlsbad, Calif.) to ensure proper pairing hadoccurred and provide a crude assessment of protein homogeneity. Toevaluate whether the recombinant mAbs retained the ability to bindcanine IL-31, mAbs were assessed for their ability to bind canine IL-31via Western Blots. Protein standards and recombinant canine IL-31 (800ng) were resolved on SDS PAGE transferred to a nitrocellulose membraneusing the Invitrogen iBlot device (Invitrogen Corp., Carlsbad, Calif.).Following transfer, membranes were washed with distilled deionized waterand blocked with 5% nonfat dried milk (NFDM) in phosphate bufferedsaline containing 0.05% tween-20 (PBST) for 1 hour at room temperature(RT). Following blocking, membranes were washed in PBST and incubatedwith either diluted supernatant from the transient expression orpurified chimeric antibodies. Binding of the chimeric antibodies wasevaluated using Goat anti-Dog IgG antibody-peroxidase conjugated (BethylLaboratories Inc., Montgomery, Tex. or Rockland, Immunochemicals, Inc.,Gilbertsville, Pa.) at a 1:5000 dilution in PBST for 1 hour at RT.Confirmation of IL-31 binding was determined by the presence of acolorimetric band (apparent molecular weight 15 kDa) corresponding tothe glycosylated form of canine IL-31 following addition of TMBsubstrate to the blot (KPL, Inc., Gaithersburg, Md.).

Chimeric mAbs showing expression from HEK 293 cells and binding to therecombinant canine IL-31 immunogen by Western blot were further analyzedfor affinity and functionality. To characterize the affinity with whichcandidate mAbs bind IL-31, surface plasmon resonance (SPR) was evaluatedusing a Biacore system (Biocore Life Sciences (GE Healthcare), Uppsala,Sweden). To avoid affinity differences associated with differentialsurface preparation that can occur when immobilizing antibodies tosurfaces; a strategy was employed where IL-31 was directly conjugated tothe surface. Immobilization was obtained by amine coupling 5 μg/mL IL-31using N-hydroxysuccinimide(NHS)/1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry.Chips were quenched with ethanolamine and the affinity with which allcandidate mAbs bound to the immobilized IL-31 was evaluated. All curveswere fit to a 1:1 model. Affinities <E-11 are below the lower limit ofquantitation of detection for the instrument.

All candidate mAbs were also evaluated for their ability to inhibitIL-31 signaling in the cell-based assay in two independent formats. Inthe co-incubation format, mAb:IL-31 complexes were pre-incubated for onehour to ensure complex formation prior to cell addition. To increase thepotential for differentiation between mAbs, a second set of experimentswere performed that lacked co-incubation and mAbs were added directly tocells for 5 minutes followed by IL-31 addition. In both cases, IL-31stimulation occurred for 5 minutes. As outlined in table 3 below,conversion of mouse monoclonals 11E12, 19D07 and 34D03 to a caninechimeric form, had little impact on their ability to bind IL-31 orinhibit cell-mediated signaling. The results also verify the correctvariable heavy and variable light chain sequences derived from eachmouse hybridoma.

TABLE 3 DH82 pSTAT Assay Co-incubation Pre-treatment Biacore AffinityAntibody IC₅₀ μg/ml IC₅₀ μg/ml K_(D) (M) Mouse 11E12 1.61 2.28 8.93E−13^(▪)Chimeric 11E12 1.48 1.57 2.68E−13 Mouse 19D07 1.76 3.46 7.24E−12*Chimeric 19D07 1.92 1.33 5.15E−13 Mouse 34D03 1.73 2.28 1.01E−12^(▪)Chimeric 34D03 1.28 1.08 4.65E−12

 Chimeric 11E12-for the heavy chain, MU-11E12-VH was paired with HC-64,and for the light chain, MU-11E12-VL was paired with kappa. *Chimeric19D07-for the heavy chain, MU-19D07-VH was paired with HC-64, and forthe light chain, MU-19D07-VL was paired with kappa.

 Chimeric 34D03-for the heavy chain, MU-34D03-VH was paired with HC-64,and for the light chain, MU-34D03-VL was paired with kappa.

Example 4 In Vivo Evaluation of Chimeric mAbs

To confirm that the inhibition of IL-31-mediated cell signaling,observed in the DH-82 assay, correlates with inhibition ofIL-31-mediated pruritus in the dog, the chimeric 11E12 monoclonalantibody described above in Table 3 (Chimeric 11E12-64) was evaluated inthe IL-31 dog pruritus model. In this model, canine IL-31, when givenintraveneously (IV) at a dose of 1 to 1.5 μg/kg, produces a fast onsetconsistent pruritic response that can be quantitated over a two hourperiod of observation. To evaluate pruritic responses, dogs were placedin single housed pens and pruritic activity measurements were performedusing video surveillance. Following an acclimation period of 1 hour,pruritic baseline scores were determined for each dog using real-timevideo surveillance using a categorical scoring system. Specifically, atconsecutive 1 minute intervals, “yes/no” decisions were made in regardsto whether pruritic behavior was displayed by each dog. Display ofpruritic behavior such actions as licking/chewing of paws, flank, and/oranal regions, scratching of flanks, neck, and/or flooring, head-shaking,and scooting of their bottom across the cage flooring was sufficient toelicit a “yes” response over the designated time interval. At the end ofthis period, the numbers of yes determinations were added together tocome up with a cumulative Pruritic Score Index (PSI). Pruritic scoreswere determined twice for each animal, with the first measurement beinga 30 minute baseline score measured immediately prior to the start ofthe test-article treatment period. After completion of each scheduledobservation period, the dogs were returned to their normal housinglocations.

To evaluate whether subcutaneous (SC) administration of chimeric11E12-64 can inhibit IL-31-mediated pruritus, a pilot study (76A03) wasperformed that included both a treated and a placebo group (N=4/group).In this study, baseline responses were performed with all 8 dogs anddogs were randomized into groups and housed based on their PSI.Importantly, each group consisted of two high responders (PSI>55) andtwo moderate responders (PSI=30 to 55). These dogs were thenadministered chimeric 11E12-64 on day 7 and IL-31 challenges wereperformed at day 8, 14 and 22. The results of this study are presentedin FIG. 8. These results demonstrate that the administration of chimeric11E12-64 resulted in a greater than 75% reduction in mean PSI for day 8and 14, relative to day 1, for chimeric 11E12-64 treated animals. Thisis in contrast to a 37-51% increase in PSI scores for untreated animals.The PSI had returned to baseline two weeks following mAb treatment,suggesting a duration of efficacy between one and two weeks for the 0.3mg/kg dose administered.

A particular challenge when assessing PSI is the day to day variationassociated with dog pruritic behavior. To help control for thisvariation, the 30 minute baseline PSI was determined for each dog, oneach day prior to IL-31 challenge. FIG. 9 shows the individual pruritcscores from doges enrolled in this study (76A60). The data in FIG. 9illustrates that the day 8 and day 14 baseline PSI was approximately 25%of the post IL-31 challenge in the chimeric 11E12-64 treated group. Thisobservation is consistent with a complete abrogation of IL-31 relatedpruritus since the baseline observation time period (0.5 h) is 25% ofthe post IL-31 observation time period (2 h). Taken together, these invivo data provide very strong evidence that; 1) the chimeric 11E12monoclonal can neutralize the ability of IL-31 to induce pruritus indogs, 2) inhibition of IL-31 mediated signaling in the cell based assaycorrelates with in vivo efficacy and 3) the parameters necessary toutilize this IL-31 model for mAb evaluation are established for theevaluation of other candidate antibodies.

Example 5 Caninization Strategy

The generation of anti-drug antibodies (ADAs) can been associated withloss of efficacy for any biotherapeutic protein including monoclonalantibodies. Comprehensive evaluation of the literature has shown thatspeciation of monoclonal antibodies can reduce the propensity for mAbsto be immunogenic although examples of immunogenic fully human mAbs andnon-immunogenic chimeric mAbs can be found. To help mitigate risksassociated with ADA formation for the mouse anti IL-31 monoclonalantibodies provided herein, a caninization strategy was employed. Thiscaninization strategy is based on identifying the most appropriatecanine germline antibody sequence for CDR grafting (FIG. 4). Followingextensive analysis of all available canine germline sequences for boththe heavy and light chain, germline candidates were selected based ontheir homology to the mouse mAbs, and the CDRs from the mouse progenitormAbs were used to replace native canine CDRs. The objective was toretain high affinity and cell-based activity using fully canineframeworks to minimize the potential of immunogenicity in vivo.Caninized mAbs were expressed and characterized for their ability tobind IL-31 via Western blotting. These results are described below inExample 8. Only mAbs that retained the ability to bind IL-31 followingcaninization were advanced for further characterization. Those mAbs thatlost the ability to bind IL-31 were systematically dissected toidentify; 1) the chain responsible for the loss of function, 2) theframework responsible for the loss of function and 3) the amino acid(s)responsible for the loss function.

Example 6 Caninization of 11E12, 19D07, and 34D03 Antibodies

Synthetic nucleotide constructs representing the caninized variableheavy and light chains of mAbs 11E12, 19D07, and 34D03 were made.Following subcloning of each variable chain into plasmids containing therespective canine heavy or kappa constant region, plasmids wereco-transfected for antibody expression in HEK 293 cells. In summary,both the 19D07 and 34D03 mAbs retained IL-31 binding upon caninization.The caninized anti-canine IL-31 variable sequences determined for eachantibody are as follows, 19D07 variable light chain (Seq ID NO: 23CAN-19D07-VL-998-1, the corresponding nucleotide sequence for which isSEQ ID NO: 46), 19D07 variable heavy chain (Seq ID NO: 29CAN-19D07-VH-400-1, the corresponding nucleotide sequence for which isSEQ ID NO: 47), 34D03 variable light chain (Seq ID NO: 25CAN-34D03-VL-998-1, the corresponding nucleotide sequence for which isSEQ ID NO: 48), and 34D03 variable heavy chain (Seq ID NO: 31CAN-34D03-VH-568-1, the corresponding nucleotide sequence for which isSEQ ID No: 49).

In contrast, the germline sequences used for the 11E12 caninizationefforts resulted in certain non-functional mAbs. With reference to FIG.10, chimeric, heterochimeric, and caninized versions of mAb 11E12 wereexpressed and characterized for their ability to bind canine IL-31 viaWestern blotting. These results demonstrated that the caninized 11E12antibody did not bind canine IL-31 (Blot #2). Also, with respect to theheterochimeras, the chimeric heavy chain paired with the caninized lightlost IL-31 binding (Blot #3), while the caninized heavy chain pairedwith the chimeric light retained IL-31 binding activity (Blot #4). Basedon the results obtained from the heterochimeras, it was deduced that thecaninized light chain was responsible for the loss of activity.

In an effort to restore the binding of caninized versions of 11E12 tocanine IL-31, the caninized light chain was modified by swappingframework sequences. FIG. 11 provides an overview of the 11E12 lightchain framework substitution work. This work identified an antibodyreplacing the canine framework II (FWII) with mouse framework II andrestoring binding to canine IL-31 (11E12 variable light chain (Seq IDNO: 20 CAN-11E12-VL-cUn-FW2, the corresponding nucleotide sequence forwhich is SEQ ID NO: 50), 11E12 variable heavy chain (Seq ID NO: 27CAN-11E12-VH-415-1, the corresponding nucleotide sequence for which isSEQ ID NO: 51)).

Further refinement of these back mutations identified an antibody with asingle arginine to leucine back mutation (R50L) in framework II couldrestore IL-31 binding via Western blot analysis (11E12 variable lightchain (Seq ID NO: 21 CAN-11E12-VL-cUn-13, the corresponding nucleotidesequence for which is SEQ ID NO: 52), 11E12 variable heavy chain (Seq IDNO: 27 CAN-11E12-VH-415-1)) (FIG. 12). Once ‘caninized’ versions of eachpotential candidate were identified the mAbs were purified and dialyzedinto PBS for further evaluation.

Table 4 summarizes the results of both the affinity measurements andcell-based inhibition data. These data demonstrate that the caninizedderivatives of both 11E12 and 34D3 both retain excellent inhibitoryactivity in the cell based assay and affinity to IL-31 as measured byBiacore. Also worth noting is the observation that while the originalcaninized 19D7 molecule retains excellent potency as measured byBiacore, the ability to inhibit cell based IL-31 signaling appearscompromised relative to its mouse progenitor. Little to no affinity losswas incurred when converting mAbs from their mouse isotype to the caninederivative.

TABLE 4 DH82 pSTAT Assay Biacore Co-incubation Pre-treatment AffinityAntibody IC₅₀ μg/ml IC₅₀ μg/ml K_(D) (M) Mouse 11E12 1.61 2.28 8.93E−13Caninized 11E12 not active not active 5.06E−07 11E12 Heterochimera 2.673.35 4.97E−12 Caninized 11E12 FW2 2.7  5.31 1.47E−10 Caninized 11E12 135.49 5.18 5.16E−12 Mouse 19D07 1.76 3.46 7.24E−12 Caninized 19D07 inc.curve inc. curve 9.23E−10 Mouse 34D03 1.73 2.28 1.01E−12 Caninized 34D032.42 2.25 2.91E−11 Variable Chain Antibody Heavy Light Caninized 11E12CAN-11E12-VH-415-1 CAN-11E12-VL-cUn-1 11E12 CAN-11E12-VH-415-1 Chimeric11E12 Heterochimera Caninized 11E12 CAN-11E12-VH-415-1CAN-11E12-VL-cUn-FW2 FW2 Caninized 11E12 CAN-11E12-VH-415-1CAN-11E12-VL-cUn-13 13 Caninized 19D07 CAN-19D07-VH-400-1CAN-19D07-VL-998-1 Caninized 34D03 CAN-34D03-VH-568-1 CAN-34D03-VL-998-1Heavy chains: All Caninized and heterochimeric forms of 11E12 includedthe V_(H) sequence of CAN-11E12-VH-415-1 (SEQ ID NO: 27) and theconstant region termed HC-64 (SEQ ID NO: 40); Caninized 19D07 includedthe V_(H) sequence of CAN-19D07-VH-400-1 (SEQ ID NO: 29) and HC-64;Caninized 34D03 included the V_(H) sequence of CAN-34D03-VH-568-1 (SEQID NO: 31) and HC-64. Light Chains: Caninized 11E12 included the V_(L)sequence of CAN-11E12-VL-cUn-1 (SEQ ID NO: 53) and the constant regiontermed kappa (SEQ ID NO: 44); Heterochimeric 11E12 included the V_(L)sequence of MU-11E12-VL (SEQ ID NO: 19) and kappa; Caninized 11E12 FW2included the V_(L) sequence of CAN-11E12-VL-cUn-FW2 (SEQ ID NO: 20) andkappa; Caninized 11E12 13 included the V_(L) sequence ofCAN-11E12-VL-cUn-13 (SEQ ID NO: 21) and kappa; Caninized 19D07 includedthe V_(L) sequence of CAN-19D07-VL-998-1 (SEQ ID NO: 23) and kappa;Caninized 34D03 included the V_(L) sequence of CAN-34D03-VL-998-1 (SEQID NO: 25) and kappa.

Example 7 Characterization of Canine IL-31 Binding to Antibodies 11E12and 34D03

To determine the amino acid residues involved with binding of canineIL-31 to antibodies 11E12 and 34D03, a mutational strategy was used thatinvolved 1) truncation of the IL-31 protein from both the N and Cterminus and 2) replacement of individual amino acids with alanine (alascan) to determine the impact on mAb binding. PCR primers were designedto amplify a canine IL-31 gene that was codon optimized for expressionin an E. coli host. The sequence of this codon-optimized canine IL-31full-length construct for E. coli expression is represented by SEQ IDNO: 55, the corresponding nucleotide sequence for which is SEQ ID NO:56. Primers were designed to amplify the full length gene and to create20 amino acid truncations of the protein moving inward from the N and Ctermini. For the purpose of these N-terminal truncations, position 1corresponded to the glycine residue immediately following the N-terminal6-His tag in the codon-optimized construct. PCR amplification productswere cloned into pET101D (Invitrogen Corp., Carlsbad, Calif.) accordingto the manufacturers protocol. The pET101D plasmid allows fusion of therecombinant protein to an N-terminal 6-His epitope tag for confirmationof expression. Sequence confirmed plasmids were used to transform BL21Star TOP10 E. coli cells (Invitrogen Corp., Carlsbad, Calif.) andexpression of the recombinant protein was induced using 1 mM Isopropylβ-D-1-thiogalactopyranoside (IPTG) under standard culture conditions.Following inductions, cells were pelleted and lysed using BacterialProtein Extraction Reagents (abbreviated B-PER, ThermoFisher ScientificInc., Rockford, Ill.). Crude lysates were subjected to SDS-PAGE andWestern blotting was carried out as described previously. All Westernblotting for mutational analysis was performed using the mouse versionsof 11E12 and 34D03 due to the availability of necessary purifiedantibodies and reagents. Each antibody was tested for its ability tobind the crude protein lysate blot representing full length andtruncated IL-31. Control blots were also probed with the anti-His mAb toconfirm expression of each protein. Proteins with an N-terminaltruncation (−20N, −40N, and −60N) all showed robust expression in E.coli and were capable of binding to 11E12 and 34D03 (FIG. 13). The aminoacid and nucleotide sequences corresponding to the −20N construct areSEQ ID NOs: 57 and 58, respectively. The amino acid and nucleotidesequences corresponding to the −40N construct are SEQ ID NOs: 59 and 60,respectively. The amino acid and nucleotide sequences corresponding tothe −60N construct are SEQ ID NOs: 61 and 62, respectively. However,full length IL-31 and proteins with C-terminal truncations (−20C, −40C,and −60C) failed to express under these conditions.

It was observed that the full length IL-31 protein was expressed verypoorly. However, the construct with the first 20 amino acids removed(−20N) from the N-terminus showed robust expression. Antibodies 11E12and 34D03 all bound to the −20N protein. Therefore, further work wascarried out using this −20 N construct. Constructs representingC-terminal truncations at positions 20-122 (MW 15.3 with his tag),20-100 (MW 12.9 with his tag), and 20-80 (MW 10.4 with his tag), weremade to assess mAb binding to these areas on the IL-31 protein. Theamino acid and nucleotide sequences corresponding to the 20-122construct are SEQ ID NOs: 63 and 64, respectively. The amino acid andnucleotide sequences corresponding to the 20-100 construct are SEQ IDNOs: 65 and 66, respectively. The amino acid and nucleotide sequencescorresponding to the 20-80 construct are SEQ ID NOs: 67 and 68,respectively. FIG. 14 shows Western blots of crude protein lysates ofthese truncated proteins that were probed with mAbs 11E12 (Blot B) and34D03 (Blot C). As shown in this Figure, mAbs 11E12 and 34D03 bound toIL-31 truncated proteins 20-122 and 20-100, but failed to bind to 20-80.These results indicated that amino acids between positions 80 and 100 ofthe canine IL-31 full-length construct of SEQ ID NO: 55 (using “SSHMA”as the N-terminus) were involved with binding of these antibodies. Thisregion corresponds to amino acid Nos. between amino acid residues 102and 122 of the canine IL-31 full-length protein sequence of SEQ ID NO:32. A control blot using the anti-His mAb (Blot A) showed that alltruncated proteins were being expressed. In addition, the pET101D-lacZprotein was used as a control to confirm the lack of non-specificbinding of mAbs to host proteins.

To further identify the amino acids in canine IL-31 involved withbinding to mAbs 11E12 and 34D03, alanine-scanning mutagenesis wasperformed according to known methods. Individual constructs were made(in the −20N plasmid) substituting alanine for each position on canineIL-31 from amino acids 76 through 122. Following sequence confirmation,protein expression was carried out and crude protein lysates weresubjected to Western blot analysis. FIG. 15 shows a summary of resultsindicating positions on canine IL-31 that, when mutated to alanine,impact binding by mAbs 11E12 and 34D03. As shown in this Figure,positions 77, 78, 81 and 85 of the full-length IL-31 construct allimpact binding of 11E12 or 34D03 antibodies. These correspond to aminoacid residues 99, 100, 103 and 107, respectively, of the canine IL-31full-length protein sequence of SEQ ID NO: 32.

To examine the impact of multiple mutations in the region of IL-31important for binding to the 11E12 and 34D03 antibodies, expressionplasmids were constructed with double (D82A, I85A) and triple (I81A,D82A, I85A) alanine substitutions. E. coli lysates expressing canineIL-31 with these double and triple mutations in addition to the −20Ncontrol were blotted with 11E12 and 34D03 antibodies (FIG. 16). It isapparent that these three amino acids on canine IL-31 are involved withrecognition of 11E12 and 34D03 as complete abrogation of binding isobserved when these sites are changed to alanine. These three aminoacids correspond to amino acid residues 103, 104 and 107 of the canineIL-31 full-length protein sequence of SEQ ID NO: 32.

In summary, truncation analysis of canine IL-31 revealed amino acidresidues (annotated in FIG. 15 between positions 80 and 122) areinvolved in binding 11E12 and 34D03 antibodies. Further, fine mutationalanalysis using alanine scanning revealed that ASP77, LYS78, ILE81,ASP82, and ILE85 of the full-length IL-31 construct all impact bindingof 11E12 or 34D03 indicating this region most likely defines the epitoperesponsible for recognition by these antibodies. Interestingly, thisregion of the human IL-31 protein was shown to be involved with bindingto the GPL subunit of its co-receptor (Le Saux S et al. Biol Chem. 2010Jan. 29; 285(5):3470-7. Epub 2009 Nov. 17). These observations, alongwith the ability of mAbs 11E12 and 34D03 to neutralize IL-31 mediatedpSTAT activity in monocytes, support the hypothesis that these mAbs bindto residues on canine IL-31 that are essential for binding of thiscytokine to its receptor, thereby inhibiting its ability to inducesignaling.

Example 8 Production of Caninized 34D03 Antibodies from GlutamineSynthetase (GS) Plasmids

The genes encoding the caninized 34D03 mAb (heavy and light chains,Table 4 above) were cloned into GS plasmids pEE 6.4 and pEE 12.4,respectively (Lonza, Basel, Switzerland). The resulting plasmids weredigested according to the manufacturer's protocol and ligated togetherto form a single mammalian expression plasmid. Each plasmid was used totransfect HEK 293 cells and expression was carried out in 20 L ofculture media. Protein was isolated from conditioned HEK medium usingProtein A affinity chromatography according to standard proteinpurification methods. Medium was loaded onto chromatographic resin andeluted by pH shift. Eluted protein was pH adjusted, dialyzed, andsterile filtered prior to use. The resulting antibody was greater than99 percent monomeric by analytical size exclusion chromatography with nohigh molecular weight aggregates observed. This antibody wassubsequently used for evaluation in the dog pruritus model to evaluatein vivo efficacy.

Example 9 Evaluation of the Caninized 34D03 Antibody in the Dog PruritusModel

The anti-pruritic activity of caninized 34D03 (CAN 34D03-65 representedby SEQ ID NO 31 (VH) paired with SEQ ID NO 25 (VL) on SEQ ID NO 42(HC-65) and SEQ ID NO 44 (LC-Kappa)) was evaluated using a canine modelof IL-31-induced pruritus. With this model, a 1.5 μg/kg intravenouschallenge dose of recombinant canine IL-31 known to induce a transientperiod of pruritic behavior in beagle dogs (IL-31 challenge, pruritusduration <24 hour) was repeatedly delivered to animals before and up to63 days after a single 1.0 mg/kg SC dose of CAN 34D03-65. At each IL-31challenge period, real-time video surveillance was used to obtain ameasure of pruritic behavior for 0.5 hours prior to cytokine delivery(pre-IL-31 baseline period) followed by a similar 2 hour measurementbeginning 20 minutes after cytokine injection (2 h post-IL-31 challengeperiod). Pruritic scores were generated at each time period underevaluation by making “yes/no” determinations as to whether a pruriticbehavior was displayed over consecutive 1 minute time-intervals (maximalpruritic score=30 for each baseline period; 120 for the post-IL-31challenge period). FIG. 17 summarizes the pruritic scores obtainedbefore and after CAN 34 D03-65 treatment, which was given on day 0 ofthe study. Seven days prior to mAb treatment, the mean post-IL-31challenge pruritic score of the dogs was 68±13 (S. E., n=4). Bycomparison, on study days 7, 14, 21, the mean post-IL-31 challengepruritic scores had lowered to 5±2, 8±4, and 9±5, respectively. Thesechanges in pruritic score between day −7 and days 7-21 represent a ≥85%decrease in overall pruritic reactivity to IL-31. The degree ofinhibition of IL-31-induced pruritus may actually have been closer to100% over this time-frame if one considers that between days 0 and 21,the 0.5 h pre-IL-31 baseline scores averaged 1.6±0.6—a level that wouldextrapolate to a pruritic score of 6-7 over a 2 h period. The pruriticreactivity of the treated dogs to exogenous IL-31 did gradually recoverover time. By day 63 post-CAN D03-65 treatment, the mean 2 h post-IL-31challenge pruritic score had increased to 57±8 or roughly 84% of thepre-mAb IL-31 challenge responses observed on day −7. Thus, in a modelof IL-31-induced pruritus, a single bolus SC injection of CAN 34D03-65did provide weeks of anti-pruritic protection to treated dogs.

Example 10 Characterization of Feline IL-31

The sequence of feline IL-31 was identified by a similarity searching ofthe feline genome with canine IL-31 using the NCBIs genome resources(www.ncbi.nlm.nih.gov). The gene representing feline IL-31 wassynthesized for optimal expression in E. coli. Expression constructswere created with full length canine and feline IL-31 genes containingan N-terminal 6-His tag for detection and purification. The felinefull-length construct used for expression in E. coli is represented bythe nucleotide sequence of SEQ ID NO: 69 and the protein sequence of SEQID NO: 70. Sequence confirmed plasmids were used to transform E. coliBL21 Star™ (Invitrogen Corp., Carlsbad, Calif.) and expression wascarried out at 30 C for 5 hours. Following lysis of cell pelletsimmunoreactive reactive protein was found to be highly enriched in theinsoluble lysate. These cell pellets were solubilized in 6M urea andpurification of the recombinant proteins was carried out underdenaturing conditions using a nickel cobalt resin (Thermo FisherScientific Inc., Rockford, Ill). Pooled eluted fractions, shown to bepositive for the presence of the His tag, were step dialyzed against 0.8M urea PBS followed by PBS, and analyzed by SDS PAGE (FIG. 18). As waspreviously observed, the yield of recombinant canine IL-31 from E. coliinduction was low. However, protein was recovered post purification thatmigrated according to expected molecular mass via SDS-PAGE.

To examine the biological activity of canine and feline IL-31 producedfrom E. coli, each protein was analyzed for its ability to induce pSTATsignaling in the DH82 cell assay. As recombinant IL-31 from mammaliancells (canine IL-31(CHO)) is highly glycosylated, it was unclear whetherthe unglycosylated form would retain biological activity. FIG. 19 showsthat feline IL-31 has comparable bioactivity to the reference referenceIL-31 produced in CHO cells.

Alanine-scanning mutagenesis of canine IL-31 defined a region within theprotein that is necessary for binding to the 11E12 and 34D03 antibodies.It was hypothesized, due to sequence conservation in this region (FIG.20), that these mAbs would cross-react with feline IL-31.

FIG. 21 shows that mAbs 11E12 and 34D03 are capable of binding to canineIL-31 (E. coli) and are also capable of cross-reacting with the felineIL-31 protein. Based on these data, speciation of the 34D03 antibody tofeline (felinization) was pursued.

Example 11 Felinization on Antibody 34D03

Similar to the caninization strategy described, appropriate germlineantibody sequences were identified from all available feline sequencesfor CDR grafting from mAb 34D03. Variable light chain (SEQ ID NO: 71FEL-34D03-VL-021-1, the corresponding nucleotide sequence for which isSEQ ID NO: 72) and variable heavy chain (SEQ ID NO: 73FEL-34D03-VH-035-1, the corresponding nucleotide sequence for which isSEQ ID NO: 74) were selected based on the highest homology to theirrespective canine frameworks in caninized 34D03. Recombinant felinized34D03 was produced using the selected variable regions joined to theirrespective constant heavy IgG1 (SEQ ID NO: 75 HC-A Feline, thecorresponding nucleotide sequence for which is SEQ ID NO: 76 GenBankaccession No. AB016710.1) and kappa constant light (SEQ ID NO: 77LC-Kappa Feline, the corresponding nucleotide sequence for which is SEQID NO: 78 GenBank accession No. AF198257.1) chain sequence. Antibody wasproduced from HEK cells and purified as previously described. FIG. 22shows the ability of feline 34D03 to neutralize pSTAT signaling with acomparable 1050 to the canine version.

Felinized 34D03 was assessed for its ability to bind both feline andcanine IL-31. FIG. 23 shows Western blots with felinized 34D03 usingpurified protein from both mammalian and E. coli sources. Conclusivebinding was observed to both canine and feline proteins indicating fullcross-reactivity of the felinized form of 34D03 and verification ofbinding to the feline protein. Taken together, these results suggest aconserved epitope on feline IL-31 may be a suitable target forinhibition of this cytokine in cats.

Example 12 Detection of IL-31 Cytokine in Dogs with Naturally OccurringAtopic Dermatitis

In the present example, the level of IL-31 protein in serum collectedfrom populations of dogs, including those with atopic dermatitis, wasevaluated using a quantitative immunoassay technique.

Serum was collected from the following populations of dogs and frozenprior to IL-31 serum measurements.

1) Twenty four purpose bred beagles (Marshall BioResources, North Rose,N.Y.) prior to and after sensitization to house dust mite allergen(Dermatophagoides farina, Greer Labs). All animals were approximately 9months in age. The two sexes were represented approximately equally.

2) Thirty flea allergic dogs (Youngs Veterinary Research Services,Turlock, Calif.) prior to flea infestation or approximately one weekafter infestation with adult cat fleas (Ctenocephalides felis). Themajority of the dogs in this colony were of mixed breed. The average agewas approximately 10.5 years. The two sexes were representedapproximately equally.

3) Eighty seven client-owned dogs with sub-clinical periodontal diseasebut otherwise determined to be in good health. Samples were collectedacross 18 US veterinary clinics. Animals were representative of the UScanine population in terms of gender and breed and were between the ageof two and five years.

4) Two hundred and twenty four client-owned animals diagnosed withchronic, non-seasonal atopic dermatitis of at least 1-year duration(based on modified Willemse's criteria, and Prelaud (Willemse T. J smallAnim Pract 1986; 27:771-778 and Prelaud et al. Revue de MedecineVeterinaire 1998; 149:1057-1064) with a minimum of “moderate itching” asassessed by the Owner, and a minimum skin lesion score of 25 on theCADESI-02) as assessed by a veterinarian. Samples were collected from 14US veterinary practices with expertise in veterinary dermatology.Approximately 75% of the dogs were purebred and ˜25% of the totalpopulation were retrievers (Labrador (17.3%) and Golden (8.2%)). Dogstended to be middle-aged (˜6 years old). The two sexes were representedapproximately equally.

A sandwich immunoassay was used to quantitate cIL-31 levels in canineserum. Serum samples were diluted 1:2 in Rexxip buffer (Gyrolab, Warren,N.J.) and run on Bioaffy 1000 nL CDs (Gyrolab) using the Gyrolab xPworkstation. cIL-31 was captured with a biotin-labeled anti-IL-31monoclonal antibody according to the present invention and detected withan Alexaflour 647 labeled anti-IL-31 monoclonal antibody according tothe present invention. Sample concentrations of cIL-31 were extrapolatedfrom an 8-point standard curve with a dynamic range of 0.013-250 ng/mLusing a 5-parmameter fit model with Gyrolab Evaluator software.

Levels of cIL-31 were detectable in serum samples of 57% of dogs withnaturally occurring atopic dermatitis (≥13 μg/mL) but were notdetectable (<13 μg/mL) in the serum from purpose-bred beagles +/−sensitized to HDM, mixed breed dogs +/− flea infestation, orclient-owned dogs with periodontal disease but otherwise considered ingood health, regardless of breed. In the dogs with naturally occurringatopic dermatitis, 53% of the samples analyzed showed serum IL-31 levelsbetween 13-1000 μg/mL, and 4% showed levels above 1000 μg/mL (Table 5).

TABLE 5 Serum IL-31 Levels in Various Canine Populations Number ofAnimals Percent of Number of with Detectable Animals Animals IL-31 withDetectable Canine Populations Evaluated in Serum^(a) IL-31 in SerumPurpose-bred beagles 24 0 0% Purpose-bred beagles 24 0 0% sensitized toHDM Mixed breed dogs - no fleas 30 0 0% Mixed breed dogs - infested 30 00% with fleas Healthy client owned 87 0 0% animals - multiple breedsNaturally occurring atopic 224 128 57% dermatitis in client ownedanimals - multiple breeds ^(a)Less than 13 pg/mL is below limits ofquantitiation.

The results of the present example demonstrate that IL-31 protein iselevated in a significant number of dogs with canine atopic dermatitis.Without wishing to be bound by any one theory, it is believed that theIL-31 pathway plays a role in the pathobiology of pruritic allergic skinconditions such as, but not limited to, canine atopic dermatitis andrepresents a novel pathway for therapeutic intervention with an IL-31antagonist, such as including, but not limited to, olacitnib and/or ananti-IL-31 antibody that specifically binds to canine IL-31.

What is claimed is:
 1. An IL-31 dog pruritus model comprising:administering canine IL-31 to dogs to produce a pruritic response;quantitatively measuring pruritic responses in the dogs which wereadministered canine IL-31; administering a candidate dog IL-31inhibitor; assessing the effectiveness of the candidate dog IL-31inhibitor in reducing prurtic behavior in the treated dogs bychallenging the dogs with canine IL-31 following the administration ofthe candidate dog IL-31 inhibitor.
 2. The IL-31 dog pruritus model ofclaim 1, wherein the canine IL-31 is recombinant canine IL-31.
 3. TheIL-31 dog pruritus model of claim 2, wherein the recombinant canineIL-31 has the amino acid sequence of SEQ ID NO:
 32. 4. The IL-31 dogpruritus model of claim 1, wherein the canine IL-31 is administeredparenterally.
 5. The IL-31 dog pruritus model of claim 1, wherein thecanine IL-31 is administered at a dose of 1 to 1.5 μg/kg.
 6. The IL-31dog pruritus model of claim 1, wherein the pruritic response is atransient response.
 7. The IL-31 dog pruritus model of claim 1, whereinthe transient pruritic response lasts less than 24 hours.
 8. The IL-31dog pruritus model of claim 1, wherein the pruritic responses in thedogs are selected from the group consisting of licking, chewing,scratching, head-shaking, and scooting.
 9. The IL-31 dog pruritus modelof claim 1, wherein pruritic behavior measurements are performed usingreal-time video surveillance using a categorical scoring system.
 10. TheIL-31 dog pruritus model of claim 9, wherein at consecutive timeintervals, “yes/no” decisions are made in regards to whether puriticbehavior was displayed by each dog.
 11. The IL-31 dog pruritus model ofclaim 10, wherein the consecutive time intervals are 1 minute intervals.12. The IL-31 dog pruritus model of claim 10, wherein at the end of adesignated observation period, the numbers of yes determinations areadded together to come up with a cumulative Pruritic Score Index (PSI).13. The IL-31 dog pruritus model of claim 12, wherein a first PSImeasurement is a baseline score measured immediately prior to the canineIL-31 challenge.
 14. The IL-31 dog pruritus model of claim 13, whereinan additional PSI measurement is determined following the canine IL-31challenge.
 15. The IL-31 dog pruritus model of claim 1, wherein thecandidate dog IL-31 inhibitor is an antibody that specifically binds tocanine IL-31.
 16. The IL-31 dog pruritus model of claim 15, wherein theantibody specifically binds to a canine IL-31 having the amino acidsequence of SEQ ID NO:
 32. 17. The IL-31 dog pruritus model of claim 16,wherein the antibody is a monoclonal antibody.
 18. The IL-31 dogpruritus model of claim 17, wherein the monoclonal antibody is caninizedor felinized.
 19. The IL-31 dog pruritus model of claim 1, wherein thecandidate dog IL-31 inhibitor is administered parenterally.